Polycomb-associated Non-Coding RNAs

ABSTRACT

This invention relates to long non-coding RNAs (lncRNAs), libraries of those lncRNAs that bind chromatin modifiers, such as Polycomb Repressive Complex 2, inhibitory nucleic acids and methods and compositions for targeting lncRNAs.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/265,104, filed on Sep. 14, 2016, which is a continuation of U.S.patent application Ser. No. 15/050,273, filed on Feb. 22, 2016, now U.S.Pat. No. 10,119,144, which is a continuation of U.S. patent applicationSer. No. 13/884,670, filed on May 10, 2013, now U.S. Pat. No. 9,328,346,which is a U.S. National Phase Application under 35 U.S.C. § 371 ofInternational Patent Application No. PCT/US2011/060493, filed on Nov.12, 2011, which claims priority to U.S. Provisional Patent ApplicationNos. 61/512,754, filed on Jul. 28, 2011, 61/425,174, filed on Dec. 20,2010, and 61/412,862, filed on Nov. 12, 2010. The entirety of each ofthe foregoing is incorporated by reference herein.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No. RO1-GM-090278 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

REFERENCE TO SEQUENCE LISTING SUBMITTED ON A COMPACT DISC

This application includes a compact disc containing a sequence listing.The sequence listing is identified on the compact disc as follows.

File Name Date of Creation Size 36708-0775008_SL.txt Jun. 16, 2021 584MBThe entire contents of the sequence listing is hereby incorporated byreference.

TECHNICAL FIELD

This invention relates to long non-coding RNAs (lncRNAs) that functionto modulate gene expression, and methods of using them or inhibitorynucleic acids that bind them, to modulate gene expression.

BACKGROUND

Transcriptome analyses have suggested that, although only 1-2% of themammalian genome is protein-coding, 70-90% is transcriptionally active(Carninci et al., 2005; Kapranov et al., 2007; Mercer et al., 2009).Ranging from 100 nt to >100 kb, these transcripts are largely unknown infunction, may originate within or between genes, and may be conservedand developmentally regulated (Kapranov et al., 2007; Guttman et al.,2009). Recent discoveries argue that a subset of these transcripts playcrucial roles in epigenetic regulation. For example, genes in the humanHOX-D locus are regulated in trans by HOTAIR RNA, produced by theunlinked HOX-C locus (Rinn et al., 2007), and during X-chromosomeinactivation, Tsix, RepA, and Xist RNAs target a chromatin modifier incis to control chromosome-wide silencing (Zhao et al., 2008).Interestingly, all four RNAs bind and regulate Polycomb RepressiveComplex 2 (PRC2), the complex that catalyzes trimethylation of histoneH3-lysine27 (H3-K27me3) (Schwartz and Pirrotta, 2008). Theseobservations support the idea that long ncRNAs are ideal for targetingchromatin modifiers to specific alleles or unique locations in thegenome (Lee, 2009) (Lee, 2010).

RNA-mediated recruitment is especially attractive for Polycomb proteins.First identified in Drosophila as homeotic regulators, Polycomb proteinsare conserved from flies to mammals and control many aspects ofdevelopment (Ringrose and Paro, 2004; Boyer et al., 2006; Lee et al.,2006; Schuettengruber et al., 2007; Pietersen and van Lohuizen, 2008;Schwartz and Pirrotta, 2008). Mammalian PRC2 contains four coresubunits, Eed, Suz12, RbAp48, and the catalytic Ezh2. In humans,aberrant PRC2 expression is linked to cancer and disease (Sparmann andvan Lohuizen, 2006; Bernardi and Pandolfi, 2007; Miremadi et al., 2007;Rajasekhar and Begemann, 2007; Simon and Lange, 2008). Despite growingrecognition of Polycomb's role in health, little is known about theirregulation in vivo. In flies, Polycomb complexes may containsequence-specific DNA-binding factors, such as Zeste, Pipsqueak (PSQ),or Pho, to help bind Polycomb-response elements (PRE) (Ringrose andParo, 2004; Schwartz and Pirrotta, 2008). By contrast, mammalianPolycomb complexes are not thought to contain such subunits. Therefore,their mechanism of recruitment to thousands of genomic locations remainspoorly understood, though PRE-like elements (Sing et al., 2009; Woo etal., 2010) and Jarid2 may facilitate binding (Li et al.; Pasini et al.;Peng et al., 2009; Shen et al., 2009). Interestingly, several PRC2subunits have potential RNA-binding motifs (Denisenko et al., 1998;Bernstein and Allis, 2005; Bernstein et al., 2006b)—a possibility borneout by postulated functional interactions between Tsix/RepA/Xist RNA andPRC2 for X-inactivation (Zhao et al., 2008) and by HOTAIR and PRC2 forHOX regulation (Rinn et al., 2007). Recent work also identified severalshort RNAs of 50-200 nt as candidate PRC2 regulators (Kanhere et al.,2010). Control of Polycomb repressive comp lex 1 (PRC1) may also involveRNA (Yap et al., 2010).

In spite of their ubiquity, the structure and function of many longncRNAs remain largely uncharacterized. Recent studies suggest that somelong ncRNAs may have a function as an epigenetic regulator/RNA cofactorin chromatin remodeling and tumor suppression. Although knockdowntechnologies employing siRNAs and shRNAs have become staples infunctional analysis of microRNAs (miRNAs) and cytoplasmically localizedmessenger RNAs (mRNAs) (4-6), these methods have been reported in someinstances to be less consistently effective for long ncRNAs localized tothe nucleus (Jepsen et al., Oligonucleotides, 14, 130-146 (2004)).

SUMMARY

A method, referred to herein as “RNA immunoprecipitation (RIP)-seq,” wasused to identify a genome-wide pool of >9,000 polycomb repressivecomplex 2 (PRC2)-interacting RNAs in embryonic stem cells (referred toherein as the “PRC2 transcriptome”). The transcriptome includesantisense, intergenic, and promoter-associated transcripts, as well asmany unannotated RNAs. A large number of transcripts occur withinimprinted regions, oncogene and tumor suppressor loci, andstem-cell-related bivalent domains. Evidence for direct RNA-proteininteractions, some via the Ezh2 subunit, is provided. Further evidenceis provided that inhibitory oligonucleotides that specifically bind tothese PRC2-interacting RNAs can successfully up-regulate gene expressionin a variety of separate and independent examples, presumably byinhibiting PRC2-associated repression. Gtl2 RNA was identifed as a PRC2cofactor that directs PRC2 to the reciprocally imprinted Dlk1 codinggene. Thus, Polycomb proteins interact with a genome-wide family ofRNAs, some of which may be used as biomarkers and therapeutic targetsfor human disease. In one aspect, the invention provides methods forpreparing a plurality of validated cDNAs complementary to a pool ofnuclear ribonucleic acids (nRNAs). The methods include providing asample comprising nuclear ribonucleic acids, e.g., a sample comprisingnuclear lysate, e.g., comprising nRNAs bound to nuclear proteins;contacting the sample with an agent, e.g., an antibody, that bindsspecifically to a nuclear protein that is known or suspected to bind tonuclear ribonucleic acids, under conditions sufficient to form complexesbetween the agent and the protein; isolating the complexes; synthesizingDNA complementary to the nRNAs to provide an initial population ofcDNAs; PCR-amplifying, if necessary, using strand-specific primers;purifying the initial population of cDNAs to obtain a purifiedpopulation of cDNAs that are at least about 20 nucleotides (nt) inlength, e.g., at least 25, 50, 75, 100, 150, 200, or 250 nt in length;sequencing at least part or substantially all of the purified populationof cDNAs; aligning reads to a reference genome and retaining only thosethat are aligned; selecting high-confidence cDNA sequences, e.g., basedon two criteria—(1) that the candidate transcript has a minimum readdensity in reads per kilobase per million reads (RPKM) terms (e.g.,above a desired threshold); and (2) that the candidate transcript isenriched in the wildtype library versus a suitable control library (suchas an IgG pulldown library or a protein-null pulldown library); therebypreparing the plurality of cDNAs.

Some examples of nuclear proteins that are known or suspected to bind tonuclear ribonucleic acids include Ezh2 (Zhao et al., Science. 2008 Oct.31; 322(5902):750-6; Khalil et al., Proc Natl Acad Sci USA. 2009 Jul.14; 106(28):11667-72. Epub 2009 Jul. 1); G9a (Nagano et al., Science.2008 Dec. 12; 322(5908):1717-20. Epub 2008 Nov. 6); and Cbx7 (Yap etal., Mol Cell. 2010 Jun. 11; 38(5):662-74.)

In some embodiments, the invention includes methods for preparing aplurality of validated cDNAs complementary to a pool of nuclearribonucleic acids (nRNAs). The methods can include providing a samplecomprising nuclear ribonucleic acids, e.g., a sample comprising nuclearlysate, e.g., comprising nRNAs bound to nuclear proteins; contacting thesample with an agent, e.g., an antibody, that binds specifically to anuclear protein that is known or suspected to bind to nuclearribonucleic acids, e.g., Ezh2, G9a, or Cbx7, under conditions sufficientto form complexes between the agent and the protein, e.g., such that thenRNAs remain bound to the proteins; isolating the complexes;synthesizing DNA complementary to the nRNAs to provide an initialpopulation of cDNAs; optionally PCR-amplifying the cDNAs usingstrand-specific primers; purifying the initial population of cDNAs toobtain a purified population of cDNAs that are at least about 20nucleotides (nt) in length, e.g., at least 25, 50, 100, 150 or 200 nt inlength;

sequencing at least part or substantially all of the purified populationof cDNAs; comparing the high-confidence sequences to a reference genome,and selecting those sequences that have a high degree of identity tosequences in the reference genome, e.g., at least 95%, 98%, or 99%identity, or that have fewer than 10, 5, 2, or 1 mismatches; andselecting those cDNAs that have (i) reads per kilobase per million reads(RPKM) above a desired threshold, and (ii) are enriched as compared to acontrol library (e.g., a protein-null library or library made from anIgG pulldown done in parallel); thereby preparing the library of cDNAs.

In some embodiments, the method is used to prepare a libraryrepresenting a transcriptome associated with the protein of interest.

In some embodiments, the agent is an antibody and isolating thecomplexes comprises immunoprecipitating the complexes. In someembodiments, the cDNAs are synthesized using strand-specific adaptors.

In some embodiments, the methods further include sequencingsubstantially all of the cDNAs.

In another aspect, the invention features libraries of cDNAscomplementary to a pool of nuclear ribonucleic acids (nRNAs) prepared bythe method of claims 1-4. In some embodiments, each of the cDNAs islinked to an individually addressable bead or area on a substrate (e.g.,a microarray).

In another aspect the invention features an inhibitory nucleic acid thatspecifically binds to, or is complementary to, an RNA that binds toPolycomb repressive complex 2 (PRC2), for example, SEQ ID NOS:1-193,049. Without being bound by a theory of invention, theseinhibitory nucleic acids are able to interfere with the binding of andfunction of PRC2, by preventing recruitment of PRC2 to a specificchromosomal locus. For example, data herein shows that a singleadministration of inhibitory nucleic acids designed to specifically binda lncRNA can stably displace not only the lncRNA, but also the PRC2 thatbinds to the lncRNA, from binding chromatin. After displacement, thefull complement of PRC2 is not recovered for up to 24 hours. Dataprovided herein also indicate that putative lncRNA binding sites forPRC2 show no conserved primary sequence motif, making it possible todesign specific inhibitory nucleic acids that will interfere with PRC2interaction with a single lncRNA, without generally disrupting PRC2interactions with other lncRNAs. Further, data provided herein supportthat lncRNA can recruit PRC2 in a cis fashion, repressing geneexpression at or near the specific chromosomal locus from which thelncRNA was transcribed, thus making it possible to design inhibitorynucleic acids that inhibit the function of PRC2 and increase theexpression of a specific target gene.

In some embodiments, the inhibitory nucleic acid is provided for use ina method of modulating expression of a “gene targeted by thePRC2-binding RNA” (e.g., an intersecting or nearby gene, as set forth inTables 1-8 below), meaning a gene whose expression is regulated by thePRC2-binding RNA. The term “PRC2-binding RNA” or “RNA that binds PRC2”is used interchangeably with “PRC2-associated RNA” and “PRC2-interactingRNA”, and refers to a lncRNA, RNA transcript or a region thereof (e.g.,a Peak as described below) that binds the PRC2 complex, directly orindirectly. Such binding may be determined by immunoprecipitationtechniques using antibodies to a component of the PRC2 complex, e.g.Ezh2. SEQ ID NOS: 1-193,049 represent murine RNA sequences containingportions that have been experimentally determined to bind PRC2 using theRIP-seq method described herein, or human RNA sequences corresponding tothese murine RNA sequences.

Such methods of modulating gene expression may be carried out in vitro,ex vivo, or in vivo. Table 8 displays genes targeted by the PRC2-bindingRNA; the SEQ ID NOS: of the PRC2-binding RNA are set forth in the samerow as the gene name. In some embodiments, the inhibitory nucleic acidis provided for use in a method of treating disease, e.g. a diseasecategory as set forth in Table 9. The treatment may involve modulatingexpression (either up or down) of a gene targeted by the PRC2-bindingRNA, preferably upregulating gene expression. The inhibitory nucleicacid may be formulated as a sterile composition for parenteraladministration. It is understood that any reference to uses of compoundsthroughout the description contemplates use of the compound inpreparation of a pharmaceutical composition or medicament for use in thetreatment of a disease. Thus, as one nonlimiting example, this aspect ofthe invention includes use of such inhibitory nucleic acids in thepreparation of a medicament for use in the treatment of disease, whereinthe treatment involves upregulating expression of a gene targeted by thePRC2-binding RNA.

Diseases, disorders or conditions that may be treated according to theinvention include cardiovascular, metabolic, inflammatory, bone,neurological or neurodegenerative, pulmonary, hepatic, kidney,urogenital, bone, cancer, and/or protein deficiency disorders. Examplesof categories of diseases are set forth in Table 9.

In a related aspect, the invention features a process of preparing aninhibitory nucleic acid that modulates gene expression, the processcomprising the step of synthesizing an inhibitory nucleic acid ofbetween 5 and 40 bases in length, optionally single stranded, thatspecifically binds, or is complementary to, an RNA sequence that hasbeen identified as binding to PRC2, optionally an RNA of any of Tables1-8 or SEQ ID NOS: 1-193,049. This aspect of the invention may furthercomprise the step of identifying the RNA sequence as binding to PRC2,optionally through the RIP-seq method described herein.

In a further aspect of the present invention a process of preparing aninhibitory nucleic acid that specifically binds to an RNA that binds toPolycomb repressive complex 2 (PRC2) is provided, the process comprisingthe step of designing and/or synthesizing an inhibitory nucleic acid ofbetween 5 and 40 bases in length, optionally single stranded, thatspecifically binds to an RNA sequence that binds to PRC2, optionally anRNA of any of Tables 1-8 or SEQ ID NOS: 1-193,049.

In some embodiments prior to synthesizing the inhibitory nucleic acidthe process further comprises identifying an RNA that binds to PRC2.

In some embodiments the RNA has been identified by a method involvingidentifying an RNA that binds to PRC2.

In some embodiments the inhibitory nucleic acid is at least 80%complementary to a contiguous sequence of between 5 and 40 bases in saidRNA sequence that binds to PRC2. In some embodiments the sequence of thedesigned and/or synthesized inhibitory nucleic acid is based on a saidRNA sequence that binds to PRC2, or a portion thereof, said portionhaving a length of from 5 to 40 contiguous base pairs.

In some embodiments the sequence of the designed and/or synthesizedinhibitory nucleic acid is based on a nucleic acid sequence that iscomplementary to said RNA sequence that binds to PRC2, or iscomplementary to a portion thereof, said portion having a length of from5 to 40 contiguous base pairs.

The designed and/or synthesized inhibitory nucleic acid may be at least80% complementary to (optionally one of at least 90%, 95%, 96%, 97%,98%, 99% or 100% complementary to) the portion of the RNA sequence towhich it binds or targets, or is intended to bind or target. In someembodiments it may contain 1, 2 or 3 base mismatches compared to theportion of the target RNA sequence or its complement respectively. Insome embodiments it may have up to 3 mismatches over 15 bases, or up to2 mismatches over 10 bases.

The inhibitory nucleic acid or portion of RNA sequence that binds toPRC2 may have a length of one of at least 8 to 40, or 10 to 50, or 5 to50, bases, e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 bases. Where theinhibitory nucleic acid is based on an RNA sequence that binds to PRC2,a nucleic acid sequence that is complementary to said RNA sequence thatbinds to PRC2 or a portion of such a sequence, it may be based oninformation about that sequence, e.g. sequence information available inwritten or electronic form, which may include sequence informationcontained in publicly available scientific publications or sequencedatabases.

Where the design and/or synthesis involves design and/or synthesis of asequence that is complementary to a nucleic acid described by suchsequence information the skilled person is readily able to determine thecomplementary sequence, e.g. through understanding of Watson-Crick basepairing rules which form part of the common general knowledge in thefield.

In the methods described above the RNA that binds to PRC2 may be, orhave been, identified, or obtained, by a method that involvesidentifying RNA that binds to PRC2.

Such methods may involve the following steps: providing a samplecontaining nuclear ribonucleic acids, contacting the sample with anagent that binds specifically to PRC2 or a subunit thereof, allowingcomplexes to form between the agent and protein in the sample,partitioning the complexes, synthesizing nucleic acid that iscomplementary to nucleic acid present in the complexes.

If necessary, the method may further comprise the steps of amplifyingthe synthesized nucleic acid, and/or purifying the nucleic acid (oramplified nucleic acid), and/or sequencing the nucleic acids soobtained, and/or filtering/analysing the nucleic acids so obtained toidentify high-probability PRC2 (or subunit thereof)-interactingtranscripts.

In one embodiment the method involves the Rip-Seq method describedherein.

In accordance with the above, in some embodiments the RNA that binds toPRC2 may be one that is known to bind PRC2, e.g. information about thesequence of the RNA and/or its ability to bind PRC2 is available to thepublic in written or electronic form allowing the design and/orsynthesis of the inhibitory nucleic acid to be based on thatinformation. As such, an RNA that binds to PRC2 may be selected fromknown sequence information and used to inform the design and/orsynthesis of the inhibitory nucleic acid.

In other embodiments the RNA that binds to PRC2 may be identified as onethat binds PRC2 as part of the method of design and/or synthesis.

In preferred embodiments design and/or synthesis of an inhibitorynucleic acid involves manufacture of a nucleic acid from startingmaterials by techniques known to those of skill in the art, where thesynthesis may be based on a sequence of an RNA (or portion thereof) thathas been selected as known to bind to Polycomb repressive complex 2.

Methods of design and/or synthesis of an inhibitory nucleic acid mayinvolve one or more of the steps of:

Identifying and/or selecting an RNA sequence that binds to PRC2;

Identifying and/or selecting a portion of an RNA sequence that binds toPRC2;

Designing a nucleic acid sequence having a desired degree of sequenceidentity or complementarity to an RNA sequence that binds to PRC2 or aportion thereof;

Synthesizing a nucleic acid to the designed sequence;

Mixing the synthesized nucleic acid with at least one pharmaceuticallyacceptable diluent, carrier or excipient to form a pharmaceuticalcomposition or medicament.

Inhibitory nucleic acids so designed and/or synthesized may be useful inmethod of modulating gene expression as described herein.

As such, the process of preparing an inhibitory nucleic acid may be aprocess that is for use in the manufacture of a pharmaceuticalcomposition or medicament for use in the treatment of disease,optionally wherein the treatment involves modulating expression of agene targeted by the RNA binds to PRC2.

In yet another aspect, the invention provides isolated nucleic acidcomprising a sequence referred to in Table 1, 2, 3, 6, and/or 7, orTable 8, or in Appendix I of U.S. Prov. Appln. No. 61/425,174 filed onDec. 20, 2010, which is not attached hereto but is incorporated byreference herein in its entirety, or a fragment comprising at least 20nt thereof, e.g., as shown in Appendix I. In some embodiments, theisolated nucleic acid is synthetic.

In a further aspect, the invention provides methods for decreasingexpression of an oncogene in a cell. In some embodiments, the methodsinclude contacting the cell with a long non-coding RNA, or PRC2-bindingfragment thereof, as referred to in Table 6 or a nucleic acid sequencethat is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% homologous to a lncRNA sequence, or PRC2-binding fragmentthereof, as referred to in Table 6. PRC2-binding fragments of murine ororthologous lncRNAs, including human lncRNA, which retain the lncRNA'sability to bind PRC2, are contemplated. In some embodiments, theoncogene is c-myc. In some embodiments, the long non-coding RNA is Pvt1.

In yet another aspect, the invention features methods for increasingexpression of a tumor suppressor in a mammal, e.g. human, in needthereof. The methods include administering to said mammal an inhibitorynucleic acid that specifically binds, or is complementary, to a humanPRC2-interacting lncRNA corresponding to a tumor suppressor locus ofTable 7, or a human lncRNA corresponding to an imprinted gene of

Table 1, and/or a human lncRNA corresponding to a growth-suppressinggene of Table 2, or a related naturally occurring lncRNA that isorthologous or at least 90%, (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical over at least 15 (e.g., at least 20, 21,25, 30, 100) nucleobases thereof, in an amount effective to increaseexpression of the tumor suppressor. Thus, it is understood that onemethod of determining human orthologous lncRNA that corresponds tomurine lncRNA is to identify a corresponding human sequence at least 90%identical to at least 15 nucleobases of the murine sequence (or at least20, 21, 25, 30, 40, 50, 60, 70, 80, 90 or 100).

In an additional aspect, the invention provides methods for inhibitingor suppressing tumor growth in a mammal, e.g. human, with cancercomprising administering to said mammal an inhibitory nucleic acid thatspecifically binds, or is complementary, to a human PRC2-interactinglncRNA corresponding to a tumor suppressor locus of Table 7, or a humanlncRNA corresponding to an imprinted gene of

Table 1, and/or a human lncRNA corresponding to a growth-suppressinggene of Table 2, or a related naturally occurring lncRNA that isorthologous or at least 90%, (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical over at least 15 (e.g., at least 20, 21,25, 30, 50, 70, 100) nucleobases thereof, in an amount effective tosuppress or inhibit tumor growth.

In another aspect, the invention features methods for treating a mammal,e.g., a human, with cancer comprising administering to said mammal aninhibitory nucleic acid that specifically binds, or is complementary, toa human lncRNA corresponding to a tumor suppressor locus of Table 7, ora human lncRNA corresponding to an imprinted gene of Table 1, and/or ahuman lncRNA corresponding to a growth-suppressing gene of

Table 2, or a related naturally occurring lncRNA that is orthologous orat least 90% (e.g.,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical over at least 15 (e.g., at least 20, 21, 25, 30, 50, 70, 100)nucleobases thereof, in a therapeutically effective amount.

In some or any embodiments, the inhibitory nucleic acid is an oligomericbase compound or oligonucleotide mimetic that hybridizes to at least aportion of the target nucleic acid and modulates its function. In someor any embodiments, the inhibitory nucleic acid is single stranded ordouble stranded. A variety of exemplary inhibitory nucleic acids areknown and described in the art. In some examples, the inhibitory nucleicacid is an antisense oligonucleotide, locked nucleic acid (LNA)molecule, peptide nucleic acid (PNA) molecule, ribozyme, siRNA,antagomirs, external guide sequence (EGS) oligonucleotide, microRNA(miRNA), small, temporal RNA (stRNA), or single- or double-stranded RNAinterference (RNAi) compounds. It is understood that the term “LNAmolecule” refers to a molecule that comprises at least one LNAmodification; thus LNA molecules may have one or more locked nucleotides(conformationally constrained) and one or more non-locked nucleotides.It is also understood that the term “LNA” includes a nucleotide thatcomprises any constrained sugar that retains the desired properties ofhigh affinity binding to complementary RNA, nuclease resistance, lack ofimmune stimulation, and rapid kinetics. Exemplary constrained sugarsinclude those listed below. Similarly, it is understood that the term“PNA molecule” refers to a molecule that comprises at least one PNAmodification and that such molecules may include unmodified nucleotidesor internucleoside linkages.

In some or any embodiments, the inhibitory nucleic acid comprises atleast one nucleotide and/or nucleoside modification (e.g., modifiedbases or with modified sugar moieties), modified internucleosidelinkages, and/or combinations thereof. Thus, inhibitory nucleic acidscan comprise natural as well as modified nucleosides and linkages.Examples of such chimeric inhibitory nucleic acids, including hybrids orgapmers, are described below.

In some embodiments, the inhibitory nucleic acid comprises one or moremodifications comprising: a modified sugar moiety, and/or a modifiedinternucleoside linkage, and/or a modified nucleotide and/orcombinations thereof. In some embodiments, the modified internucleosidelinkage comprises at least one of: alkylphosphonate, phosphorothioate,phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate,carbonate, phosphate triester, acetamidate, carboxymethyl ester, orcombinations thereof. In some embodiments, the modified sugar moietycomprises a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxymodified sugar moiety, a 2′-O-alkyl modified sugar moiety, or a bicyclicsugar moiety. Other examples of modifications include locked nucleicacid (LNA), peptide nucleic acid (PNA), arabinonucleic acid (ANA),optionally with 2′-F modification, 2′-fluoro-D-Arabinonucleic acid(FANA), phosphorodiamidate morpholino oligomer (PMO), ethylene-bridgednucleic acid (ENA), optionally with 2′-O,4′-C-ethylene bridge, andbicyclic nucleic acid (BNA). Yet other examples are described belowand/or are known in the art.

In some embodiments, the inhibitory nucleic acid is 5-40 bases in length(e.g., 12-30, 12-28, 12-25). The inhibitory nucleic acid may also be10-50, or 5-50 bases length. For example, the inhibitory nucleic acidmay be one of any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 bases inlength. In some embodiments, the inhibitory nucleic acid is doublestranded and comprises an overhang (optionally 2-6 bases in length) atone or both termini. In other embodiments, the inhibitory nucleic acidis double stranded and blunt-ended. In some embodiments, the inhibitorynucleic acid comprises or consists of a sequence of bases at least 80%or 90% complementary to, e.g., at least 5, 10, 15, 20, 25 or 30 basesof, or up to 30 or 40 bases of, the target RNA, or comprises a sequenceof bases with up to 3 mismatches (e.g., up to 1, or up to 2 mismatches)over 10, 15, 20, 25 or 30 bases of the target RNA.

Thus, the inhibitory nucleic acid can comprise or consist of a sequenceof bases at least 80% complementary to at least 10 contiguous bases ofthe target RNA, or at least 80% complementary to at least 15, or 15-30,or 15-40 contiguous bases of the target

RNA, or at least 80% complementary to at least 20, or 20-30, or 20-40contiguous bases of the target RNA, or at least 80% complementary to atleast 25, or 25-30, or 25-40 contiguous bases of the target RNA, or atleast 80% complementary to at least 30, or 30-40 contiguous bases of thetarget RNA, or at least 80% complementary to at least 40 contiguousbases of the target RNA. Moreover, the inhibitory nucleic acid cancomprise or consist of a sequence of bases at least 90% complementary toat least 10 contiguous bases of the target RNA, or at least90%complementary to at least 15, or 15-30, or 15-40 contiguous bases ofthe target RNA, or at least 90% complementary to at least 20, or 20-30,or 20-40 contiguous bases of the target RNA, or at least 90%complementary to at least 25, or 25-30, or 25-40 contiguous bases of thetarget RNA, or at least 90% complementary to at least 30, or 30-40contiguous bases of the target RNA, or at least 90% complementary to atleast 40 contiguous bases of the target RNA. Similarly, the inhibitorynucleic acid can comprise or consist of a sequence of bases fullycomplementary to at least 5, 10, or 15 contiguous bases of the targetRNA.

Complementarity can also be referenced in terms of the number ofmismatches in complementary base pairing, as noted above. Thus, theinhibitory nucleic acid can comprise or consist of a sequence of baseswith up to 3 mismatches over 10 contiguous bases of the target RNA, orup to 3 mismatches over 15 contiguous bases of the target RNA, or up to3 mismatches over 20 contiguous bases of the target RNA, or up to 3mismatches over 25 contiguous bases of the target RNA, or up to 3mismatches over 30 contiguous bases of the target RNA. Similarly, theinhibitory nucleic acid can comprise or consist of a sequence of baseswith up to 2 mismatches over 10 contiguous bases of the target RNA, orup to 2 mismatches over 15 contiguous bases of the target RNA, or up to2 mismatches over 20 contiguous bases of the target RNA, or up to 2mismatches over 25 contiguous bases of the target RNA, or up to 2mismatches over 30 contiguous bases of the target RNA. Similarly, thethe inhibitory nucleic acid can comprise or consist of a sequence ofbases with one mismatch over 10, 15, 20, 25 or 30 contiguous bases ofthe target RNA.

As such, in some embodiments the inhibitory nucleic acid comprises orconsists of a sequence of bases about 5 to 40, or 10 to 50, or 5 to 50bases in length, comprising a base sequence at least 80% complementaryto (optionally one of at least 90%, 95%, 96%, 97%, 98%, 99% or 100%complementary to) a contiguous sequence of at least 5 to 40 bases(optionally one of at least 10, 15, 20, 25 or 30 bases, or one of 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50 bases) of the target lncRNA. Thus, in someembodiments the inhibitory nucleic acid may comprise or consist of asequence of at least 5 to 40, or 5 to 50,or 10 to 50, bases (optionallyone of at least 10, 15, 20, 25 or 30 bases, or one of 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, or 50 bases) having at least 80% identity to (optionally oneof at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to) acontiguous sequence of bases of the same length of an antisense nucleicacid that is completely complementary in sequence to the target lncRNA.In some embodiments the sequence of the inhibitory nucleic acid maycontain 1, 2 or 3 mismatches in complementary base pairing compared tothe target lncRNA sequence, over 10, 15, 20, 25 or 30 bases (optionallyone of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29 or 30 bases) of the target lncRNA.

In some or any embodiments, the inhibitory nucleic acid is 5 to 40, or10 to 50 bases in length (e.g., 12-30, 12-28, 12-25, 5-25, or 10-25,bases in length), and comprises a sequence of bases with up to 3mismatches in complementary base pairing over 15 bases, or up to 2mismatches over 10 bases.

In some embodiments, the cell is a cancer cell, e.g., a tumor cell, invitro or in vivo, e.g., in a subject. In other embodiments, the cell isa stem cell that is contacted with the inhibitory nucleic acid,PRC2-binding lncRNA, or fragment thereof, ex vivo, for example toenhance pluripotency, enhance differentiation, or induce the stem cellto differentiate to a particular cell type, e.g. nerve, neuron,dopaminergic neuron, muscle, skin, heart, kidney, liver, lung,neuroendocrine, retinal, retinal pigment epithelium, pancreatic alpha orbeta cells, hematopoietic, chondrocyte, bone cells and/or blood cells(e.g., T-cells, B-cells, macrophages, erythrocytes, platelets, and thelike).

In some embodiments, the gene is Nkx2-1 (also known as Titf1). In someembodiments, the long non-coding (antisense) RNA to Nkx2-1 is on mouseChromosome 12, approximately bp 57,636,100 to 57,638,650, likelyoverlapping the Nkx2-1 promoter, including AK14300; or NR_003367.1. Inhumans, a similar antisense transcript occurs in the human NKX2-1 locus(likely overlapping, if not coincident, with Human gene BX161496; Chr14:bp 36,988,521-36,991,722, in human genome assembly version GRCh37/hg19).Nkx2-1 is BOTH a tumor suppressor and an oncogene. Early on, it isrequired to form the tumor; but later on, its expression is lost andthat loss correlates with a bad prognosis. So the lncRNA targetingNkx2-1 has at least two uses: it can be administered itself to blockcancer formation; or later on, its expression can be reduced to drive upexpression of NKX2-1. In humans, NKX2-1 is frequently amplified ormutated in lung adenocarcinomas and has been directly linked to lungoncogenesis. It is described as a proto-oncogene in driving initialcancer development, but at the same time, its loss of expression iseventually associated with bad prognosis. Therefore, in someembodiments, the promoter-associated antisense transcript isadministered to a subject, e.g., a subject with cancer, e.g., lungadenocarcinoma, and/or introduced into tumor cells to thereby reduceexpression of NKX2-1 in patients with amplified NKX2-1 expression.Alternatively, in subjects (e.g., subjects with cancer, e.g., lungadenocarcinoma) with poor prognosis who have lost NKX2-1 expression, aninhibitory RNA, such as an LNA molecule, could be introduced toantagonize the PRC2-interacting antisense transcript and restartexpression of the NKX2-1 gene.

In an additional aspect, the invention provides methods for enhancingpluripotency of a stem cell. The methods include contacting the cellwith a long non-coding RNA, or PRC2-binding fragment thereof, asreferred to in Table 3 or a nucleic acid sequence that is at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to alncRNA sequence, or PRC2-binding fragment thereof, as referred to inTable 3. PRC2-binding fragments of murine or orthologous lncRNAs,including human lncRNA, are contemplated in the aforementioned method.

In a further aspect, the invention features methods for enhancingdifferentiation of a stem cell, the method comprising contacting thecell with an inhibitory nucleic acid that specifically binds, or iscomplementary, to a long non-coding RNA as referred to in Table 3 and 4.

In some embodiments, the stem cell is an embryonic stem cell. In someembodiments, the stem cell is an iPS cell or an adult stem cell.

In an additional aspect, the invention provides sterile compositionsincluding an inhibitory nucleic acid that specifically binds to or is atleast 90% complementary to (e.g., at least 5, 10, 15, 20, 25 or 30 basesof, or up to 30 or 40 bases of) a lncRNA of Table 1, 2, 6, or 7, orTable 8, or a related naturally occurring lncRNA at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least 15(e.g., at least 20, 21, 25, 30, 100) nucleobases of an lncRNA of Table1, 2, 6, or 7, or Table 8, for parenteral administration. In someembodiments, the inhibitory nucleic acid is selected from the groupconsisting of antisense oligonucleotides, ribozymes, external guidesequence (EGS) oligonucleotides, siRNA compounds, micro RNAs (miRNAs);small, temporal RNAs (stRNA), and single- or double-stranded RNAinterference (RNAi) compounds. In some embodiments, the RNAi compound isselected from the group consisting of short interfering RNA (siRNA); ora short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa);and small activating RNAs (saRNAs).

In some embodiments, the antisense oligonucleotide is selected from thegroup consisting of antisense RNAs, antisense DNAs, chimeric antisenseoligonucleotides, and antisense oligonucleotides.

In some embodiments, the inhibitory nucleic acid comprises one or moremodifications comprising: a modified sugar moiety, a modifiedinternucleoside linkage, a modified nucleotide and/or combinationsthereof. In some embodiments, the modified internucleoside linkagecomprises at least one of: alkylphosphonate, phosphorothioate,phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate,carbonate, phosphate triester, acetamidate, carboxymethyl ester, orcombinations thereof. In some embodiments, the modified sugar moietycomprises a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxymodified sugar moiety, a 2′-O-alkyl modified sugar moiety, or a bicyclicsugar moiety. Other examples of modifications include locked nucleicacid (LNA), peptide nucleic acid (PNA), arabinonucleic acid (ANA),optionally with 2′-F modification, 2′-fluoro-D-Arabinonucleic acid(FANA), phosphorodiamidate morpholino oligomer (PMO), ethylene-bridgednucleic acid (ENA), optionally with 2′-O,4′-C-ethylene bridge, andbicyclic nucleic acid (BNA). Yet other examples are described belowand/or are known in the art.

PRC2-binding fragments of any of the RNA set forth in the sequencelisting as summarized below are contemplated. In some aspects, thefragments may recruit PRC2 and enhance PRC2 activity, thereby repressinggene expression, while in other instances the fragments may interferewith PRC2 activity by masking the lncRNA-binding sites on PRC2. Inparticular, the invention features uses of fragments of the RNA below tomodulate expression of any of the genes set forth in Tables 1-8, for usein treating a disease, disorder, condition or association described inany of the categories set forth in Table 9 (whether in the “oppositestrand” column or the “same strand” column).

Moreover, inhibitory nucleic acids that specifically bind to any of theRNA set forth in the sequence listing as summarized below, SEQ ID NOS:1-193,049, are also contemplated. In particular, the invention featuresuses of these inhibitory nucleic acids to upregulate expression of anyof the genes set forth in the Tables 1-8, for use in treating a disease,disorder, condition or association described in any of the categoriesset forth in Table 9 (whether in the “opposite strand” column or the“same strand” column of Table 8); upregulations of a set of genesgrouped together in any one of the categories is contemplated. Evidenceis provided herein that such inhibitory nucleic acids increasedexpression of mRNA corresponding to the gene by at least about 50% (i.e.150% of normal or 1.5-fold), or by about 2-fold to about 5-fold. In someembodiments it is contemplated that expression may be increased by atleast about 15-fold, 20-fold, 30-fold, 40-fold, 50-fold or 100-fold, orany range between any of the foregoing numbers. In other experiments,increased mRNA expression has been shown to correlate to increasedprotein expression.

A summary of the sequences in the sequence listing is set forth below.

TABLE Start SEQ End SEQ Organism ID NO: ID NO: 1 Mus musculus 1 49 2 Musmusculus 50 9836 3 Mus musculus 9837 10960 4 Mus musculus 10961 11620 5Mus musculus 11621 12052 6 Mus musculus 12053 12267 7 Mus musculus 1226812603 1 Homo sapiens 12604 12632 2 Homo sapiens 12633 19236 3 Homosapiens 19237 20324 4 Homo sapiens 20325 20956 5 Homo sapiens 2095721194 6 Homo sapiens 21195 21337 7 Homo sapiens 21338 21582 8 Musmusculus Peaks 21583 124436 8 Homo sapiens Peaks 124437 190716 8 Musmusculus Peaks 190717 190933 8 Homo sapiens Peaks 190934 191086 8 H.sapiens Peak Ex. 7 191087 8 M. musculus Peak Ex. 7 191088 2 Homo sapiens191089 192873 1 Homo sapiens 192874 192885 3 Homo sapiens 192886 1929064 Homo sapiens 192907 192916 5 Homo sapiens 192917 192979 6 Homo sapiens192980 193006 7 Homo sapiens 193007 193049

The SEQ ID number refers to the RNA that associates (binds) with PRC2(i.e., the RNA against which inhibitory nucleic acids would bedirected). Each of (a) the reference genes described in the tables, (b)the Prc2-binding transcripts or Peaks (i.e., smaller regions of RNA thatbind to PRC2) that target (modulate expression of) these genes, and (c)the inhibitory nucleic acids that specifically bind to, or arecomplementary to, the PRC2-binding transcripts or Peaks, mayconveniently be grouped into any one of these categories, represented bynumbers in Table 9 as follows:

Diseases are marked by category numbers 11, 14, 15, 17, 21, 24, 26, 42,44, 49, 58, 69, 82, 103, 119, 120, 126, 143, 163, 167, 172, 177, 182,183, 184, 187, 191, 196, 200, 203, 204, 212, any one of 300-323, or anyone of 400-643.

Other functional groups are marked by category numbers 10, 12, 13, 16,18, 19, 20, 22, 23, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 43, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 121, 122, 123, 124, 125, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 164, 165, 166, 168, 169, 170, 171, 173, 174, 175, 176,178, 179, 180, 181, 185, 186, 188, 189, 190, 192, 193, 194, 195, 197,198, 199, 201, 202, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215,216, 217, or 218.

Category No. Name 10 actin cytoskeleton organization 11 Acute myeloidleukemia 12 Adherens junction 13 Adipocytokine signaling pathway 14aging 15 Alzheimer's disease 16 Amino sugar and nucleotide sugarmetabolism 17 Amyotrophic lateral sclerosis (ALS) 18 angiogenesis 19Apoptosis 20 Arginine and proline metabolism 21 Arrhythmogenic rightventricular cardiomyopathy (ARVC) 22 Axon guidance 23 B cell receptorsignaling pathway 24 Basal cell carcinoma, also in category 644 25 Basaltranscription factors 26 Bladder cancer, also in category 644 27 bloodcoagulation 28 blood vessel development 29 bone development 30 Calciumsignaling pathway 31 Cardiac muscle contraction 32 cation channelactivity 33 cell adhesion 34 cell cycle 35 Cell cycle 36 cell motion 37cell surface receptor linked signal transduction 38 cellular response tostress 39 channel activity 40 Chemokine signaling pathway 41 cholesterolmetabolic process 42 Chronic myeloid leukemia 43 Citrate cycle (TCAcycle) 44 Colorectal cancer 45 Complement and coagulation cascades 46cytokine activity 47 cytoskeletal protein binding 48 cytosol 49 Dilatedcardiomyopathy 50 DNA binding 51 DNA repair 52 DNA replication 53 DNAreplication 54 Drug metabolism 55 embryonic morphogenesis 56 endocytosis57 Endocytosis 58 Endometrial cancer 59 endoplasmic reticulum 60 ErbBsignaling pathway 61 extracellular region 62 eye development 63 Fattyacid metabolism 64 Fructose and mannose metabolism 65 G-protein coupledreceptor protein signaling pathway 66 gamete generation 67 Gap junction68 gene silencing by miRNA 69 Glioma 70 glucose metabolic process 71Glycolysis/Gluconeogenesis 72 Golgi apparatus 73 growth factor activity74 GTPase regulator activity 75 heart development 76 Hedgehog signalingpathway 77 Hematopoietic cell lineage 78 hemopoiesis 79 hemopoietic orlymphoid organ development 80 histone modification 81 Huntington'sdisease 82 Hypertrophic cardiomyopathy (HCM) 83 immune response 84immune system development 85 inflammatory response 86 Insulin signalingpathway 87 intracellular signaling cascade 88 ion channel activity 89ion transport 90 Jak-STAT signaling pathway 91 learning or memory 92leukocyte activation 93 Leukocyte transendothelial migration 94 limbdevelopment 95 locomotory behavior 96 Long-term potentiation 97 lungdevelopment 98 lysosome 99 Lysosome 100 MAPK signaling pathway 101MAPKKK cascade 102 Melanogenesis 103 Melanoma 104 Mismatch repair 105mitochondrion 106 mitochondrion organization 107 mTOR signaling pathway108 muscle tissue development 109 ncRNA metabolic process 110 neurondevelopment 111 Neurotrophin signaling pathway 112 Non-small cell lungcancer, also in category 644 113 Notch signaling pathway 114 nucleolus115 Oocyte meiosis 116 oxidation reduction 117 Oxidative phosphorylation118 p53 signaling pathway 119 Pancreatic cancer, also in category 644120 Parkinson's disease 121 Pathways in cancer, also in category 644 122phosphatase activity 123 phosphoprotein phosphatase activity 124positive regulation of cellular biosynthetic process 125 PPAR signalingpathway 126 Prostate cancer, also in category 644 127 Proteasome 128protein amino acid dephosphorylation 129 protein folding 130 proteinkinase activity 131 protein serine/threonine kinase activity 132 Purinemetabolism 133 Pyrimidine metabolism 134 Ras protein signal transduction135 Regulation of actin cytoskeleton 136 Regulation of autophagy 137regulation of cell death, also in category 644 138 regulation of cellproliferation, also in category 644 139 regulation of cell size 140regulation of protein ubiquitination 141 regulation of Ras proteinsignal transduction 142 regulation of transcription 143 Renal cellcarcinoma, also in category 644 144 response to hypoxia 145 response tosteroid hormone stimulus 146 response to virus 147 ribosome 148 RNAdegradation 149 RNA processing 150 RNA splicing, via transesterificationreactions 151 secretion 152 skeletal system development 153 skeletalsystem morphogenesis 154 Small cell lung cancer, also in category 644155 small GTPase regulator activity 156 spermatogenesis 157 Sphingolipidmetabolism 158 spliceosome 159 Spliceosome 160 stem cell differentiation161 Steroid biosynthesis 162 synapse 163 Systemic lupus erythematosus164 T cell activation 165 T cell receptor signaling pathway 166 TGF-betasignaling pathway 167 Thyroid cancer, also in category 644 168 Toll-likereceptor signaling pathway 169 transcription activator activity 170transcription factor activity 171 translation 172 Type II diabetesmellitus 173 Ubiquitin mediated proteolysis 174 Vascular smooth musclecontraction 175 vasculature development 176 VEGF signaling pathway 177Viral myocarditis 178 Wnt signaling pathway 179 amino-acid biosynthesis180 ank repeat 181 bromodomain 182 Cardiomyopathy 183 cataract 184charcot-marie-tooth disease 185 cytokine 186 cytokine receptor 187deafness 188 disease mutation 189 egf-like domain 190 endosome 191epilepsy 192 glycoprotein 193 growth factor 194 Growth factor binding195 growth factor receptor 196 Ichthyosis 197 Immunoglobulin domain 198ionic channel 199 leucine-rich repeat 200 leukodystrophy 201 methylation202 methyltransferase 203 neurodegeneration 204 neuropathy 205 nucleus206 obesity 207 protein phosphatase 208 protein phosphatase inhibitor209 Oncogene (including proto-oncogenes), also in category 644 210Secreted 211 serine/threonine-specific protein kinase 212 systemic lupuserythematosus 213 transmembrane 214 transmembrane protein 215 tumorsuppressor, also in category 644 216 tyrosine-protein kinase 217 ublconjugation pathway 218 wd repeat 300 Downregulated in Bladder cancer,also in category 644 301 Downregulated in Leukemia, also in category 644302 Downregulated in Brain cancer, also in category 644 303Downregulated in Breast cancer, also in category 644 304 Downregulatedin Cervical cancer, also in category 644 305 Downregulated in Coloncancer, also in category 644 306 Downregulated in Esophageal cancer,also in category 644 307 Downregulated in Gastric cancer, also incategory 644 308 Downregulated in Head and Neck cancer, also in category644 309 Downregulated in Renal cancer, also in category 644 310Downregulated in Liver cancer, also in category 644 311 Downregulated inLung cancer, also in category 644 312 Downregulated in Lymphoma, also incategory 644 313 Downregulated in Melanoma, also in category 644 314Downregulated in Multiple Myeloma, also in category 644 315Downregulated in Ovarian cancer, also in category 644 316 Downregulatedin Pancreatic cancer, also in category 644 317 Downregulated in Prostatecancer, also in category 644 318 Downregulated in Sarcoma, also incategory 644 319 Downregulated in Non-melanoma skin cancer, also incategory 644 320 Downregulated in Uterine cancer, also in category 644321 Downregulated in Mesothelioma, also in category 644 322Downregulated in Adrenal cancer, also in category 644 323 Downregulatedin Parathyroid cancer, also in category 644 400 Upregulated in Clearcell sarcoma of kidney, also in category 644 401 Upregulated in Acutelung injury 402 Upregulated in Acute megakaryoblastic leukemia, also incategory 644 403 Upregulated in Acute myelocytic leukemia, also incategory 644 404 Upregulated in Acute pancreatitis unspecified 405Upregulated in Adenocarcinoma of esophagus, also in category 644 406Upregulated in Adenocarcinoma of lung, also in category 644 407Upregulated in Adenoma of small intestine, also in category 644 408Upregulated in Adenovirus infection 409 Upregulated in AIDS withencephalitis 410 Upregulated in Alcohol poisoning 411 Upregulated inAlexander disease 412 Upregulated in alpha-1-Antitrypsin deficiency 413Upregulated in Alzheimer's disease 414 Upregulated in Anaplasticoligoastrocytoma, also in category 644 415 Upregulated in Androgeninsensitivity syndrome 416 Upregulated in Astrocytoma, also in category644 417 Upregulated in Atrophy-muscular 418 Upregulated in Autoimmunehepatitis 419 Upregulated in Bacterial infection 420 Upregulated inBarrett's esophagus 421 Upregulated in Carcinoma in situ of smallintestin, also in category 644e 422 Upregulated in Cardiomyopathy 423Upregulated in Chronic granulomatous disease 424 Upregulated in Chroniclymphocytic leukemia 425 Upregulated in Chronic obstructive airwaydisease 426 Upregulated in Chronic polyarticular juvenile rheumatoidarthritis 427 Upregulated in Cirrhosis of liver 428 Upregulated inCocaine dependence 429 Upregulated in Complex dental caries 430Upregulated in Crohn's disease 431 Upregulated in Decompensated cardiacfailure 432 Upregulated in Dehydration 433 Upregulated in Dilatedcardiomyopathy 434 Upregulated in Dilated cardiomyopathy secondary toviral myocarditis 435 Upregulated in Epithelial proliferation 436Upregulated in Escherichia coli infection of the central nervous system437 Upregulated in Essential thrombocythemia 438 Upregulated inExhaustion due to excessive exertion 439 Upregulated in Familialhypophosphatemic bone disease 440 Upregulated in Fracture 441Upregulated in Fracture of femur 442 Upregulated in Generalized ischemicmyocardial dysfunction 443 Upregulated in Glioblastoma, also in category644 444 Upregulated in Hamman-Rich syndrome 445 Upregulated inHelicobacter pylori gastrointestinal tract infection 446 Upregulated inHepatitis C 447 Upregulated in HIV infection 448 Upregulated inHuntington's disease 449 Upregulated in Hypercholesterolemia 450Upregulated in Hypertrophy 451 Upregulated in Idiopathicthrombocytopenic purpura 452 Upregulated in Infection by Yersiniaenterocolitica 453 Upregulated in Infertility due to azoospermia 454Upregulated in Injury of heart 455 Upregulated in ISM-In situ melanomaof skin 456 Upregulated in Leber's amaurosis 457 Upregulated in Livercarcinoma, also in category 644 458 Upregulated in Macular degeneration459 Upregulated in Malignant lymphoma, also in category 644 460Upregulated in Malignant neoplasm of cervix uteri, also in category 644461 Upregulated in Malignant neoplasm of duodenum, also in category 644462 Upregulated in Malignant neoplasm of prostate, also in category 644463 Upregulated in Malignant neoplasm of stomach, also in category 644464 Upregulated in Malignant neoplasm of testis, also in category 644465 Upregulated in Malignant tumor of colon, also in category 644 466Upregulated in Multiple benign melanocytic nevi 467 Upregulated inNephropathy-diabetic 468 Upregulated in Non-insulin dependent diabetesmellitus 469 Upregulated in Nutritional deficiency 470 Upregulated inObstructive sleep apnea 471 Upregulated in Oligodendroglioma, also incategory 644 472 Upregulated in Papillary thyroid carcinoma, also incategory 644 473 Upregulated in Parkinson disease 474 Upregulated inPorcine nephropathy 475 Upregulated in Pre-eclampsia 476 Upregulated inPrimary cardiomyopathy 477 Upregulated in Primary open angle glaucoma478 Upregulated in Primary pulmonary hypoplasia 479 Upregulated inPseudomonas infection 480 Upregulated in Pulmonary emphysema 481Upregulated in Pulmonary hypertension 482 Upregulated in Renal disorderassociated with type II diabetes mellitus 483 Upregulated in Retinaldamage 484 Upregulated in Retinitis pigmentosa 485 Upregulated inRheumatoid arthritis 486 Upregulated in Squamous cell carcinoma, also incategory 644 487 Upregulated in Squamous cell carcinoma of lung, also incategory 644 488 Upregulated in Status epilepticus 489 Upregulated inSystemic infection 490 Upregulated in Thrombocytopenia 491 Upregulatedin Thymic carcinoma, also in category 644 492 Upregulated inTransitional cell carcinoma, also in category 644 493 Upregulated inTransitional cell carcinoma in situ, also in category 644 494Upregulated in Ulcerative colitis 495 Upregulated in Uterine fibroids496 Upregulated in Ventilator-associated lung injury 497 Upregulated inVentricular hypertrophy 498 Upregulated in Ventricular hypertrophy (&[left]) 499 Upregulated in Vitamin A deficiency 500 Downregulated inClear cell sarcoma of kidney, also in category 644 501 Downregulated inAcute lung injury 502 Downregulated in Acute megakaryoblastic leukemia,also in category 644 503 Downregulated in Acute myelocytic leukemia,also in category 644 504 Downregulated in Acute pancreatitis unspecified505 Downregulated in Adenocarcinoma of esophagus, also in category 644506 Downregulated in Adenocarcinoma of lung, also in category 644 507Downregulated in Adenoma of small intestine, also in category 644 508Downregulated in Adenovirus infection 509 Downregulated in AIDS withencephalitis 510 Downregulated in Alcohol poisoning 511 Downregulated inAlexander disease 512 Downregulated in alpha-1-Antitrypsin deficiency513 Downregulated in Alzheimer's disease 514 Downregulated in Anaplasticoligoastrocytoma 515 Downregulated in Androgen insensitivity syndrome516 Downregulated in Astrocytoma, also in category 644 517 Downregulatedin Atrophy-muscular 518 Downregulated in Autoimmune hepatitis 519Downregulated in Bacterial infection 520 Downregulated in Barrett'sesophagus 521 Downregulated in Carcinoma in situ of small intestine,also in category 644 522 Downregulated in Cardiomyopathy 523Downregulated in Chronic granulomatous disease 524 Downregulated inChronic lymphocytic leukemia 525 Downregulated in Chronic obstructiveairway disease 526 Downregulated in Chronic polyarticular juvenilerheumatoid arthritis 527 Downregulated in Cirrhosis of liver 528Downregulated in Cocaine dependence 529 Downregulated in Complex dentalcaries 530 Downregulated in Crohn's disease 531 Downregulated inDecompensated cardiac failure 532 Downregulated in Dehydration 533Downregulated in Dilated cardiomyopathy 534 Downregulated in Dilatedcardiomyopathy secondary to viral myocarditis 535 Downregulated inEpithelial proliferation 536 Downregulated in Escherichia coli infectionof the central nervous system 537 Downregulated in Essentialthrombocythemia 538 Downregulated in Exhaustion due to excessiveexertion 539 Downregulated in Familial hypophosphatemic bone disease 540Downregulated in Fracture 541 Downregulated in Fracture of femur 542Downregulated in Generalized ischemic myocardial dysfunction 543Downregulated in Glioblastoma, also in category 644 544 Downregulated inHamman-Rich syndrome 545 Downregulated in Helicobacter pylorigastrointestinal tract infection 546 Downregulated in Hepatitis C 547Downregulated in HIV infection 548 Downregulated in Huntington's disease549 Downregulated in Hypercholesterolemia 550 Downregulated inHypertrophy 551 Downregulated in Idiopathic thrombocytopenic purpura 552Downregulated in Infection by Yersinia enterocolitica 553 Downregulatedin Infertility due to azoospermia 554 Downregulated in Injury of heart555 Downregulated in ISM-In situ melanoma of skin, also in category 644556 Downregulated in Leber's amaurosis 557 Downregulated in Livercarcinoma, also in category 644 558 Downregulated in Maculardegeneration 559 Downregulated in Malignant lymphoma, also in category644 560 Downregulated in Malignant neoplasm of cervix uteri, also incategory 644 561 Downregulated in Malignant neoplasm of duodenum, alsoin category 644 562 Downregulated in Malignant neoplasm of prostate,also in category 644 563 Downregulated in Malignant neoplasm of stomach,also in category 644 564 Downregulated in Malignant neoplasm of testis,also in category 644 565 Downregulated in Malignant tumor of colon, alsoin category 644 566 Downregulated in Multiple benign melanocytic nevi567 Downregulated in Nephropathy-diabetic 568 Downregulated inNon-insulin dependent diabetes mellitus 569 Downregulated in Nutritionaldeficiency 570 Downregulated in Obstructive sleep apnea 571Downregulated in Oligodendroglioma 572 Downregulated in Papillarythyroid carcinoma 573 Downregulated in Parkinson disease 574Downregulated in Porcine nephropathy 575 Downregulated in Pre-eclampsia576 Downregulated in Primary cardiomyopathy 577 Downregulated in Primaryopen angle glaucoma 578 Downregulated in Primary pulmonary hypoplasia579 Downregulated in Pseudomonas infection 580 Downregulated inPulmonary emphysema 581 Downregulated in Pulmonary hypertension 582Downregulated in Renal disorder associated with type II diabetesmellitus 583 Downregulated in Retinal damage 584 Downregulated inRetinitis pigmentosa 585 Downregulated in Rheumatoid arthritis 586Downregulated in Squamous cell carcinoma, also in category 644 587Downregulated in Squamous cell carcinoma of lung, also in category 644588 Downregulated in Status epilepticus 589 Downregulated in Systemicinfection 590 Downregulated in Thrombocytopenia 591 Downregulated inThymic carcinoma, also in category 644 592 Downregulated in Transitionalcell carcinoma, also in category 644 593 Downregulated in Transitionalcell carcinoma in situ, also in category 644 594 Downregulated inUlcerative colitis 595 Downregulated in Uterine fibroids 596Downregulated in Ventilator-associated lung injury 597 Downregulated inVentricular hypertrophy 598 Downregulated in Ventricular hypertrophy (&[left]) 599 Downregulated in Vitamin A deficiency 600 is associated withBone diseases 601 is associated with Cancer diseases, also in category644 602 is associated with Cardiovascular diseases 603 is associatedwith Connective tissue disorder diseases 604 is associated withDermatological diseases 605 is associated with Developmental diseases606 is associated with Ear, Nose, Throat diseases 607 is associated withEndocrine diseases 608 is associated with Gastrointestinal diseases 609is associated with Hematological diseases 610 is associated withImmunological diseases 611 is associated with Metabolic diseases 612 isassociated with multiple diseases 613 is associated with Musculardiseases 614 is associated with Neurological diseases 615 is associatedwith Nutritional diseases 616 is associated with Ophthamologicaldiseases 617 is associated with Other diseases 618 is associated withPsychiatric diseases 619 is associated with Renal diseases 620 isassociated with Respiratory diseases 621 is associated with Skeletaldiseases 622 is decreased in Bone diseases 623 is decreased in Cancerdiseases, also in category 644 624 is decreased in Cardiovasculardiseases 625 is decreased in Connective tissue disorder diseases 626 isdecreased in Dermatological diseases 627 is decreased in Developmentaldiseases 628 is decreased in Ear, Nose, Throat diseases 629 is decreasedin Endocrine diseases 630 is decreased in Gastrointestinal diseases 631is decreased in Hematological diseases 632 is decreased in Immunologicaldiseases 633 is decreased in Metabolic diseases 634 is decreased inmultiple diseases 635 is decreased in Muscular diseases 636 is decreasedin Neurological diseases 637 is decreased in Nutritional diseases 638 isdecreased in Ophthamological diseases 639 is decreased in Other diseases640 is decreased in Psychiatric diseases 641 is decreased in Renaldiseases 642 is decreased in Respiratory diseases 643 is decreased inSkeletal diseases 644 is involved in cancer

Thus, in various aspects, the invention features inhibitory nucleicacids that specifically bind to any of the RNA sequences of any ofTables 1-8, for use in modulating expression of a group of referencegenes that fall within any one or more of the categories set forth inthe tables, and for treating the corresponding diseases, disorders orconditions in any one or more of the categories set forth in Table 9(which sets forth the diseases associated with each reference gene).

In another aspect, the invention also features inhibitory nucleic acidsthat specifically bind, or are complementary, to any of the RNAsequences of SEQ ID NOS: 124437 to 190716, or 190934 to 191086, or191087 (human Peaks), or SEQ ID NOS: 21583 to 124436, or 190717 to190933, or 191088 (mouse Peaks), set forth in Table 8, whether in the“opposite strand” column or the “same strand” column. In someembodiments, the inhibitory nucleic acid is provided for use in a methodof modulating expression of a gene targeted by the PRC2-binding RNA(e.g., an intersecting or nearby gene, as set forth in Tables 1-8below). Such methods may be carried out in vitro, ex vivo, or in vivo.In some embodiments, the inhibitory nucleic acid is provided for use inmethods of treating disease, e.g. as described in Table 9 below. Thetreatments may involve modulating expression (either up or down) of agene targeted by the PRC2-binding RNA, preferably upregulating geneexpression. In some embodiments, the inhibitory nucleic acid isformulated as a sterile composition for parenteral administration. Thereference genes targeted by these RNA sequences are set forth in Table 8and are grouped according to categories 1-643 in Table 9. Thus, in oneaspect the invention describes a group of inhibitory nucleic acids thatspecifically bind, or are complementary to, a group of RNA sequences,either transcripts or Peaks, in any one of categories 1-643. Inparticular, the invention features uses of such inhibitory nucleic acidsto upregulate expression of any of the reference genes set forth inTable 8, for use in treating a disease, disorder, condition orassociation described in any of the categories set forth in Table 9(e.g., any one or more of category numbers 11, 14, 15, 17, 21, 24, 26,42, 44, 49, 58, 69, 82, 103, 119, 120, 126, 143, 163, 167, 172, 177,182, 183, 184, 187, 191, 196, 200, 203, 204, 212, 300-323, and/or400-643).

By way of nonlimiting example, category 45 (Complement and coagulationcascades) includes reference genes selected from the group consisting ofTFPI, F2, F2R, CD46, PROS1, SERPINE1, A2M, C1S, C3AR1, BDKRB1, C1R,SERPING1, BDKRB2, F5, C8G, THBD, and/or PLAU (gene IDs 7035, 2147, 2149,4179, 5627, 5054, 2, 716, 719, 623, 715, 710, 624, 2153, 733, 7056, and5328, respectively). In turn, each of TFPI, F2, F2R, CD46, PROS1,SERPINE1, A2M, C1S, C3AR1, BDKRB1, C1R, SERPING1, BDKRB2, F5, C8G, THBD,and/or PLAU are targeted by PRC2-binding RNA having the SEQ ID NOsdisplayed in the applicable row of Table 8. For example, TFPI SEQ ID NOsinclude 13245[F], 155228[F], 155229[F], 155230[F], 155231[F], 155232[F],155233[F], 155234[F], 155235[F], 155236[F], 155237[F], 155238[F],155239[F], 155240[F], 155241[F], 155242[F], 155243[F], 155244[F],155245[F], 155246[F], 155247[F], 155248[F], 155249[F], 155250[F],155251[F], 155252[F], 155255[F], 155256[F], 13245[66912], 155237[-806],879[F], 68709[F], 68710[F], 68711[F], 68712[F], 68713[F], 68714[F],68715[F], 68716[F], 68717[F], 68718[F], 68719[F], 68720[F], 68721[F],68722[F], 68723[F], 68724[F], 68725[F], 68726[F], 68727[F], 68728[F],68729[F], 68730[F], 68731[F], 68732[F], 68733[F], 68734[F], 68735[F],68736[F], 68737[F], 68738[F], 68739[F], 68740[F], 68741[F], 68742[F],68743[F], 68744[F], 68745[F], 68746[F], 68747[F], 68748[F], 68749[F],and/or 68713[-245] according to Table 8. The group of inhibitory nucleicacids selected from the group consisting of inhibitory nucleic acidsthat specifically bind to, or are complementary to, any one of these SEQID NOS: that are listed in Table 8 as targeting refGenes TFPI, F2, F2R,CD46, PROS1, SERPINE1, A2M, C1S, C3AR1, BDKRB1, C1R, SERPING1, BDKRB2,F5, C8G, THBD, and/or PLAU are contemplated for use in any of thecompositions and methods described herein, including but not limited touse in treating a disease of category 45 (Complement and coagulationcascades), the treatment involving modulation of any of the refGenesTFPI, F2, F2R, CD46, PROS1, SERPINE1, A2M, C1S, C3AR1, BDKRB1, C1R,SERPING1, BDKRB2, F5, C8G, THBD, and/or PLAU. Similarly, inhibitorynucleic acids that specifically bind to, or are complementary to, genesin category 643 (“is decreased in Skeletal disease”) are contemplatedfor use in any of the compositions and methods described herein,including but not limited to use in treating Skeletal disease.Inhibitory nucleic acids that specifically bind to, or are complementaryto, genes in the categories that are also part of category 644 (involvedin cancer) are contemplated for use in any of the compositions andmethods described herein, including but not limited to use in treatingcancer.

In various aspects, the invention further features inhibitory nucleicacids that bind to the RNA sequence between two or more Peaks thatcorrespond to chromosomal coordinates that are near each other, e.g.within 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1kb, or2kb of each other, and that are preferably associated with (i) the samereference gene in Table 9 or (ii) the same UCSC transcript in Table 9.For example, the invention features inhibitory nucleic acids thatspecifically bind, or are complementary to, a fragment of any of the RNAtranscripts of SEQ ID NOS: 1 to 21582 or or 191089 to 193049, saidfragment about 2000, about 1750, about 1500, or about 1250 nucleotidesin length, or preferably about 1000, about 750, about 500, about 400,about 300 nucleotides in length, or more preferably about 200, about150, or about 100 nucleotides in length, wherein the fragment of RNAcomprises a stretch of at least five (5) consecutive nucleotides withinany of SEQ ID NOS: 124437 to 190716, or 190934 to 191086, or 191087(human Peaks), or SEQ ID NOS: 21583 to 124436, or 190717 to 190933, or191088 (mouse Peaks), or the reverse complement of any of the cDNAsequences of Appendix I of U.S. Prov. Appl. No. 61/425,174 filed on Dec.20, 2010, which is not attached hereto but is incorporated by referenceherein in its entirety. In exemplary embodiments the fragment of RNAcomprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 consecutivenucleotides within any of SEQ ID NOS: 124437 to 190716, or 190934 to191086, or 191087 (human Peaks), or SEQ ID NOS: 21583 to 124436, or190717 to 190933, or 191088 (mouse Peaks), or the reverse complement ofany of the cDNA sequences of Appendix I of U.S. Prov. Appl. No.61/425,174 filed on Dec. 20, 2010.

In some or any embodiments, the inhibitory nucleic acids are, e.g.,about 5 to 40, or 10 to 50 bases, or 5 to 50 bases in length. In someembodiments, the inhibitory nucleic acid comprises or consists of asequence of bases at least 80% or 90% complementary to, e.g., at least5, 10, 15, 20, 25 or 30 bases of, or up to 30 or 40 bases of, the targetRNA (i.e., any one of SEQ ID NOS: 1-193,049), or comprises a sequence ofbases with up to 3 mismatches (e.g., up to 1, or up to 2 mismatches)over 10, 15, 20, 25 or 30 bases of the target RNA.

Thus, as noted above, the inhibitory nucleic acid can comprise orconsist of a sequence of bases at least 80% complementary to at least10, or 10-30, or 10-40 contiguous bases of the target RNA, or at least80% complementary to at least 15, or 15-30, or 15-40 contiguous bases ofthe target RNA, or at least 80% complementary to at least 20, or 20-30,or 20-40 contiguous bases of the target RNA, or at least 80%complementary to at least 25, or 25-30, or 25-40 contiguous bases of thetarget RNA, or at least 80% complementary to at least 30, or 30-40contiguous bases of the target RNA, or at least 80% complementary to atleast 40 contiguous bases of the target RNA. Moreover, the inhibitorynucleic acid can comprise or consist of a sequence of bases at least 90%complementary to at least 5, or 5-30, or 5-40 contiguous bases of thetarget RNA, or at least 90% complementary to at least 10, or 10-30, or10-40 contiguous bases of the target RNA, or at least 90% complementaryto at least 15, or 15-30, or 15-40 contiguous bases of the target RNA,or at least 90% complementary to at least 20, or 20-30, or 20-40contiguous bases of the target RNA, or at least 90% complementary to atleast 25, or 25-30, or 25-40 contiguous bases of the target RNA, or atleast 90% complementary to at least 30, or 30-40 contiguous bases of thetarget RNA, or at least 90% complementary to at least 40 contiguousbases of the target RNA. Similarly, the inhibitory nucleic acid cancomprise or consist of a sequence of bases fully complementary to atleast 5, 10, or 15 contiguous bases of the target RNA. It is understoodthat some additional non-complementary bases may be included. It isunderstood that inhibitory nucleic acids that comprise such sequences ofbases as described may also comprise other non-complementary bases. Forexample, an inhibitory nucleic acid can be 20 bases in total length butcomprise a 15 base portion that is fully complementary to 15 bases ofthe target RNA. Similarly, an inhibitory nucleic acid can be 20 bases intotal length but comprise a 15 base portion that is at least 80%complementary to 15 bases of the target RNA.

Complementarity can also be referenced in terms of the number ofmismatches in complementary base pairing, as noted above. Thus, theinhibitory nucleic acid can comprise or consist of a sequence of baseswith up to 3 mismatches over 10 contiguous bases of the target RNA, orup to 3 mismatches over 15 contiguous bases of the target RNA, or up to3 mismatches over 20 contiguous bases of the target RNA, or up to 3mismatches over 25 contiguous bases of the target RNA, or up to 3mismatches over 30 contiguous bases of the target RNA. Similarly, theinhibitory nucleic acid can comprise or consist of a sequence of baseswith up to 2 mismatches over 10 contiguous bases of the target RNA, orup to 2 mismatches over 15 contiguous bases of the target RNA, or up to2 mismatches over 20 contiguous bases of the target RNA, or up to 2mismatches over 25 contiguous bases of the target RNA, or up to 2mismatches over 30 contiguous bases of the target RNA. Similarly, thethe inhibitory nucleic acid can comprise or consist of a sequence ofbases with one mismatch over 10, 15, 20, 25 or 30 contiguous bases ofthe target RNA.

In some or any of the embodiments of inhibitory nucleic acids describedherein (e.g. in the summary, detailed description, or examples ofembodiments) or the processes for designing or synthesizing them, theinhibitory nucleic acids may optionally exclude (a) any one or more ofthe specific inhibitory nucleic acids made or actually disclosed (i.e.specific chemistry, single or double stranded, specific modifications,and specific base sequence), set forth in the following SEQ ID NOS.;and/or (b) the base sequence of any one or more of the inhibitorynucleic acids of (a); and/or (c) the group of inhibitory nucleic acidsthat specifically bind or are complementary to the same specific portionof RNA (a stretch of contiguous bases) as any one or more of theinhibitory nucleic acids of (a); as disclosed in any one or more of thefollowing publications: as target HOTAIR RNA (Rinn et al., 2007), Tsix,RepA, or Xist RNAs ((Zhao et al., 2008) [SEQ ID NOS: 194206-194210], or(Sarma et al., 2010) [SEQ ID NOS: 194217-194226]or (Zhao et al., 2010)[SEQ ID NOS: 194227-194228] or (Prasanath, et al., 2005) [SEQ ID NOS:194213-194216] or (Shamovsky, et al., 2006) [SEQ ID NO: 194212] or(Mariner, et al., 2008) [SEQ ID NOS: 194211] or (Sunwoo, et al., 2008)or (Bernard, et al., 2010) [SEQ ID NOS: 194229]; or as targeting shortRNAs of 50-200 nt that are identified as candidate PRC2 regulators(Kanhere et al., 2010); or (Kuwabara et al., US 2005/0226848) [SEQ IDNOS: 194230-194231] or (Li et al., US 2010/0210707) [SEQ ID NOS:194232-194267] or (Corey et al., 7,709,456) [SEQ ID NOS: 194268-194285]or (Mattick et al., WO 2009/124341) or (Corey et al., US 2010/0273863)[SEQ ID NOS: 194286-194305], or (Wahlstedt et al., US 2009/0258925) [SEQID NOS: 193140-193206], or BACE: US 2009/0258925 [SEQ ID NOS:193140-193206]; ApoAl: US 2010/0105760/EP235283 [SEQ ID NOS:193207-193379], P73, p53, PTEN, WO 2010/065787 A2/EP2370582 [SEQ ID NOS:193380-193425]; SIRT1: WO 2010/065662 A2/EP09831068 [SEQ ID NOS:193426-193472]; VEGF: WO 2010/065671 A2/EP2370581 [SEQ ID NOS:193473-193483]; EPO: WO 2010/065792 A2/EP09831152 [SEQ ID NOS:193484-193492]; BDNF: WO2010/093904 [SEQ ID NOS: 193493-193503], DLK1:WO 2010/107740 [SEQ ID NOS: 193504-193510]; NRF2NFE2L2: WO 2010/107733[SEQ ID NOS: 193511-193518]; GDNF: WO 2010/093906 [SEQ ID NOS:193519-193556]; SOX2, KLF4, Oct3A/B, “reprogramming factors: WO2010/135329 [SEQ ID NOS: 193557-193573]; Dystrophin: WO 2010/129861 [SEQID NOS: 193574-193605]; ABCA1, LCAT, LRP1, ApoE, LDLR, Apo1; WO2010/129799 [SEQ ID NOS: 193606-193884]; HgF: WO 2010/127195 [SEQ IDNOS: 193885-193889]; TTP/Zfp36: WO 2010/129746 [SEQ ID NOS:193890-193904]; TFE3, IRS2: WO 2010/135695 [SEQ ID NOS: 193905-193919];RIG1, MDA5, IFNA1: WO 2010/138806 [SEQ ID NOS: 193920-193958]; PON1: WO2010/148065 [SEQ ID NOS: 193959-193965]; Collagen: WO/2010/148050 [SEQID NOS: 193966-193998]; Dyrk1A, Dscr1, “Down Syndrome Gene”:WO/2010/151674 [SEQ ID NOS: 193999-194022]; TNFR2: WO/2010/151671 [SEQID NOS: 194023-194029]; Insulin: WO/2011/017516 [SEQ ID NOS:194030-194039]; ADIPOQ: WO/2011/019815 [SEQ ID NOS: 194040-194064];CHIP: WO/2011/022606 [SEQ ID NOS: 194065-194074]; ABCB1: WO/2011/025862[SEQ ID NOS: 194075-194082]; NEUROD1, EUROD1, HNF4A, MAFA, PDX, KX6,“Pancreatic development gene”: WO/2011/085066 [SEQ ID NOS:194083-194115]; MBTPS1: WO/2011/084455 [SEQ ID NOS: 194116-194119];SHBG: WO/2011/085347 [SEQ ID NOS: 194120-194133]; IRF8: WO/2011/082409[SEQ ID NOS: 194134-194137]; UCP2: WO/2011/079263 [SEQ ID NOS:194138-194148]; HGF: WO/2011/079261 [SEQ ID NOS: 194149-194156]; GH:WO/2011/038205 [SEQ ID NOS: 194157-194161]; IQGAP: WO/2011/031482 [SEQID NOS: 194162-194166]; NRF1: WO/2011/090740 [SEQ ID NOS:194167-194172]; P63: WO/2011/090741 [SEQ ID NOS: 194173-194176];RNAseH1: WO/2011/091390 [SEQ ID NOS: 194177-194184]; ALOX12B:WO/2011/097582 [SEQ ID NOS: 194185-194189]; PYCR1: WO/2011/103528 [SEQID NOS: 194190-194193]; CSF3: WO/2011/123745 [SEQ ID NOS:194194-194198]; FGF21: WO/2011/127337 [SEQ ID NOS: 194199-194205]; ofwhich each of the foregoing is incorporated by reference in its entiretyherein. In some or any embodiments, optionally excluded from theinvention are inhibitory nucleic acids that specifically bind to, or arecomplementary to, any one or more of the following regions: Nucleotides1-932 of SEQ ID NO: 193208; Nucleotides 1-1675 of SEQ ID NO: 193386;Nucleotides 1-518 of SEQ ID NO: 193387; Nucleotides 1-759 of SEQ ID NO:193388; Nucleotides 1-25892 of SEQ ID NO: 193389; Nucleotides 1-279 ofSEQ ID NO: 193390; Nucleotides 1-1982 of SEQ ID NO: 193391; Nucleotides1-789 of SEQ ID NO: 193392; Nucleotides 1-467 of SEQ ID NO: 193393;Nucleotides 1-1028 of SEQ ID NO: 193427; Nucleotides 1-429 of SEQ ID NO:193428; Nucleotides 1-156 of SEQ ID NO: 193429; Nucleotides 1-593 of SEQID NO: 193430; Nucleotides 1-643 of SEQ ID NO: 193475; Nucleotides 1-513of SEQ ID NO: 193476; Nucleotides 1-156 of SEQ ID NO: 193486;Nucleotides 1-3175 of SEQ ID NO: 193494; Nucleotides 1-1347 of SEQ IDNO: 193506; Nucleotides 1-5808 of SEQ ID NO: 193513; Nucleotides 1-237of SEQ ID NO: 193520; Nucleotides 1-1246 of SEQ ID NO: 193521;Nucleotides 1-684 of SEQ ID NO: 193522; Nucleotides 1-400 of SEQ ID NO:193553; Nucleotides 1-619 of SEQ ID NO: 193554;Nucleotides 1-813 of SEQID NO: 193555; Nucleotides 1-993 of SEQ ID NO: 193560; Nucleotides 1-401of SEQ ID NO: 193560; Nucleotides 1-493 of SEQ ID NO: 193561;Nucleotides 1-418 of SEQ ID NO: 193562; Nucleotides 1-378 of SEQ ID NO:193576; Nucleotides 1-294 of SEQ ID NO: 193577; Nucleotides 1-686 of SEQID NO: 193578; Nucleotides 1-480 of SEQ ID NO: 193579; Nucleotides 1-501of SEQ ID NO: 193580; Nucleotides 1-1299 of SEQ ID NO: 193613;Nucleotides 1-918 of SEQ ID NO: 193614; Nucleotides 1-1550 of SEQ ID NO:193615; Nucleotides 1-329 of SEQ ID NO: 193616; Nucleotides 1-1826 ofSEQ ID NO: 193617; Nucleotides 1-536 of SEQ ID NO: 193618; Nucleotides1-551 of SEQ ID NO: 193619; Nucleotides 1-672 of SEQ ID NO: 193620;Nucleotides 1-616 of SEQ ID NO: 193621; Nucleotides 1-471 of SEQ ID NO:193622; Nucleotides 1-707 of SEQ ID NO: 193623; Nucleotides 1-741 of SEQID NO: 193624; Nucleotides 1-346 of SEQ ID NO: 193625; Nucleotides 1-867of SEQ ID NO: 193626; Nucleotides 1-563 of SEQ ID NO: 193627;Nucleotides 1-970 of SEQ ID NO: 193892; Nucleotides 1-1117 of SEQ ID NO:193893; Nucleotides 1-297 of SEQ ID NO: 193894; Nucleotides 1-497 of SEQID NO: 193907; Nucleotides 1-1267 of SEQ ID NO: 193923; Nucleotides1-586 of SEQ ID NO: 193924; Nucleotides 1-741 of SEQ ID NO: 193925;Nucleotides 1-251 of SEQ ID NO: 193926; Nucleotides 1-681 of SEQ ID NO:193927; Nucleotides 1-580 of SEQ ID NO: 193928; Nucleotides 1-534 of SEQID NO: 193960; Nucleotides 1-387 of SEQ ID NO: 193969; Nucleotides 1-561of SEQ ID NO: 193970; Nucleotides 1-335 of SEQ ID NO: 193971;Nucleotides 1-613 of SEQ ID NO: 193972; Nucleotides 1-177 of SEQ ID NO:193973; Nucleotides 1-285 of SEQ ID NO: 193974; Nucleotides 1-3814 ofSEQ ID NO: 194001; Nucleotides 1-633 of SEQ ID NO: 194002; Nucleotides1-497 of SEQ ID NO: 194003; Nucleotides 1-545 of SEQ ID NO: 194004;Nucleotides 1-413 of SEQ ID NO: 194306; Nucleotides 1-413 of SEQ ID NO:194307; Nucleotides 1-334 of SEQ ID NO: 194308; Nucleotides 1-582 of SEQID NO: 194309; Nucleotides 1-416 of SEQ ID NO: 194310; Nucleotides1-3591 of SEQ ID NO: 194311; Nucleotides 1-875 of SEQ ID NO: 194312;Nucleotides 1-194 of SEQ ID NO: 194313; Nucleotides 1-2074 of SEQ ID NO:194314; Nucleotides 1-1237 of SEQ ID NO: 194315; Nucleotides 1-4050 ofSEQ ID NO: 194316; Nucleotides 1-1334 of SEQ ID NO: 194317; Nucleotides1-1235 of SEQ ID NO: 194318; Nucleotides 1-17,964 of SEQ ID NO: 194319;Nucleotides 1-50,003 of SEQ ID NO: 194320; Nucleotides 1-486 of SEQ IDNO: 194321; Nucleotides 1-494 of SEQ ID NO: 194322; Nucleotides 1-1992of SEQ ID NO: 194323; Nucleotides 1-1767 of SEQ ID NO: 194324;Nucleotides 1-1240 of SEQ ID NO: 194325; Nucleotides 1-3016 of SEQ IDNO: 194326; Nucleotides 1-1609 of SEQ ID NO: 194327; Nucleotides 1-312of SEQ ID NO: 194328; Nucleotides 1-243 of SEQ ID NO: 194329;Nucleotides 1-802 of SEQ ID NO: 194330; Nucleotides 1-514 of SEQ ID NO:194331; Nucleotides 1-936 of SEQ ID NO: 194332; Nucleotides 1-1075 ofSEQ ID NO: 194333; Nucleotides 1-823 of SEQ ID NO: 194334; Nucleotides1-979 of SEQ ID NO: 194335; Nucleotides 1-979 of SEQ ID NO: 194336;Nucleotides 1-288 of SEQ ID NO: 194337; Nucleotides 1-437 of SEQ ID NO:194338; Nucleotides 1-278 of SEQ ID NO: 194339; Nucleotides 1-436 of SEQID NO: 194340; Nucleotides 1-1140 of SEQ ID NO: 194341; Nucleotides1-2082 of SEQ ID NO: 194342; Nucleotides 1-380 of SEQ ID NO: 194343;Nucleotides 1-742 of SEQ ID NO: 194344; Nucleotides 1-4246 of SEQ ID NO:194345.

In some or any embodiments, one or more of the murine RNA sequences ofTable 3 may be excluded. In some or any embodiments, one or more of thehuman RNA sequences of Table 3 may be excluded. In some or anyembodiments, one or more of the murine RNA sequences of Table 4 may beexcluded. In some or any embodiments, one or more of the human RNAsequences of Table 4 may be excluded. In some or any embodiments, one ormore of the murine RNA sequences of Table 5 may be excluded. In some orany embodiments, one or more of the human RNA sequences of Table 5 maybe excluded.

In some or any of the embodiments of inhibitory nucleic acids describedherein, or processes for designing or synthesizing them, the inhibitorynucleic acids will upregulate gene expression and may specifically bindor specifically hybridize or be complementary to a PRC2-binding RNA thatis transcribed from the same strand as a protein-coding reference gene.The inhibitory nucleic acid may bind to a region of the PRC2-binding RNAthat originates within or overlaps an intron, exon, intron-exonjunction, 5′ UTR, 3′ UTR, a translation initiation region, or atranslation termination region of a protein-coding sense-strand of areference gene (refGene).

In some or any of the embodiments of inhibitory nucleic acids describedherein, or processes for designing or syntheisizing them, the inhibitorynucleic acids will upregulate gene expression and may specifically bindor specifically hybridize or be complementary to a PRC2-binding RNA thattranscribed from the opposite strand (antisense-strand) compared to aprotein-coding reference gene.

The inhibitory nucleic acids described herein may be modified, e.g.comprise a modified sugar moiety, a modified internucleoside linkage, amodified nucleotide and/or combinations thereof. In addition, theinhibitory nucleic acids can exhibit one or more of the followingproperties: do not induce substantial cleavage or degradation of thetarget RNA; do not cause substantially complete cleavage or degradationof the target RNA; do not activate the RNAse H pathway; do not activateRISC; do not recruit any Argonaute family protein; are not cleaved byDicer; do not mediate alternative splicing; are not immune stimulatory;are nuclease resistant; have improved cell uptake compared to unmodifiedoligonucleotides; are not toxic to cells or mammals; may have improvedendosomal exit; do interfere with interaction of lncRNA with PRC2,preferably the Ezh2 subunit but optionally the Suz12, Eed, RbAp46/48subunits or accessory factors such as Jarid2; do decrease histoneH3-lysine27 methylation and/or do upregulate gene expression.

In some or any of the embodiments of inhibitory nucleic acids describedherein, or processes for designing or synthesizing them, the inhibitorynucleic acids may optionally exclude those that bind DNA of a promoterregion, as described in Kuwabara et al., US 2005/0226848 or Li et al.,US 2010/0210707 or Corey et al., U.S. Pat. NO. 7,709,456 or Mattick etal., WO 2009/124341, or those that bind DNA of a 3′ UTR region, asdescribed in Corey et al., US 2010/0273863.

Inhibitory nucleic acids that are designed to interact with RNA tomodulate gene expression are a distinct subset of base sequences fromthose that are designed to bind a DNA target (e.g., are complementary tothe underlying genomic DNA sequence from which the RNA is transcribed).

Also described herein are methods for the use of Locked Nucleic Acids(LNA) molecules for targeting nuclear long noncoding RNA, an RNAsubclass that has been less amenable to traditional knockdown techniques(Jepsen et al., Oligonucleotides, 14, 130-146 (2004); Khalil et al.,PNAS 106(28)11675-11680 (2009)). As described herein, LNA molecules canbe used to displace lncRNAs from cognate binding sequences (i.e. cognatebinding partners), e.g., chromosomes, or PRC2, with fast kinetics.

Thus, in one aspect, the present invention provides locked nucleic acid(LNA) molecules that are complementary to and bind specifically to longnoncoding RNAs (lncRNAs).

In another aspect, the invention features methods for dissociating(e.g., disrupting binding of or decreasing binding affinity for) a longnoncoding RNA (lncRNA) from its cognate binding partner. The methodsinclude contacting the lncRNA with a locked nucleic acid (LNA) moleculethat is complementary to and binds specifically to the lncRNA.

In some embodiments, the lncRNA is a large intergenic non-coding RNA(lincRNA), a promoter associated short RNA (PASR), an endogenousantisense RNA, or an RNA that binds a chromatin modifier, e.g., aPolycomb complex, e.g., Polycomb repressive complex 2.

In some embodiments, the lncRNA is localized to the nucleus.

In some embodiments, the LNA molecule is complementary to a region ofthe lncRNA comprising a known RNA localization motif.

In some embodiments, the LNA molecule comprises at least one non-lockednucleotide. Such LNA molecules may have one or more locked nucleotidesand one or more non-locked nucleotides. It is understood that the term“LNA” includes a nucleotide that comprises any constrained sugar thatretains the desired properties of high affinity binding to complementaryRNA, nuclease resistance, lack of immune stimulation, and rapidkinetics. Exemplary constrained sugars include those listed below.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1M show one embodiment of the RIP-seq methods as describedherein, and analysis of pilot libraries.

FIG 1A is an exemplary RIP-seq schematic.

FIG. 1B is a pair of images showing the results of Western blot analysis(right panel) of Ezh2 protein in wildtype (WT) and Ezh2−/− ES cells andCoomassie staining (left panel) demonstrating equal loading.

FIG. 1C is an image showing the results of a preparatory agarose gel forRIP product size selection.

FIG. 1D is a table showing pilot library statistics for WT and controllibraries for an equivalent number of cells (column 2), reads afterfiltering using criteria shown in FIG. 7 (column 3), and distinct readsafter removing duplicates and repetitive elements (column 4).

FIG. 1E is a table giving the CCs of indicated libraries in pairwisecomparisons against the original WT library.

FIG. 1F is a line graph showing the cumulative frequency of WT readsmapping to elements with indicated genome copy numbers.

FIG. 1G is a pie chart showing the relative frequencies of variousrepeats in the WT library. Elements repeated >10 times per genomeaccounted for <20% of all reads. Simple repeats accounted for 85.714%and LINEs, SINEs, LTRs, low-complexity repeats, and satellitesrepresented 4.881%, 4.130%, 2.636%, 2.487%, and 0.002% (not shown onchart), respectively.

FIG. 1H is a graph showing alignments of distinct WT pilot reads to themouse X-chromosome. The number of reads per 100-kb window for bothunique and repetitive elements are plotted from centromere (CEN) todistal telomere (TELO). 100-kilobase windows are nonoverlapping andconsecutive. Reads were normalized such that those mapping to ‘n’locations were counted as 1/n^(th) of a read at each location. Chr,chromosome. Dark grey, forward strand; light grey, reverse strand.

FIG. 1I is a graph showing a zoom-in of the X-inactivation centerlisting pilot WT reads. The Ezh2−/− library is depleted of these reads.Freq≥3 reads shown. *, ncRNA.

FIG. 1J shows a pair of graphs. On the top is a graph showing alignmentsof distinct WT pilot reads to the mouse chromosome 12. The number ofreads per 100-kb window for both unique and repetitive elements areplotted from centromere (CEN) to distal telomere (TELO). 100-kilobasewindows are nonoverlapping and consecutive. Reads were normalized suchthat those mapping to ‘n’ locations were counted as 1/nth of a read ateach location. Chr, chromosome. Dark grey, forward strand; light grey,reverse strand. On the bottom is a graph showing a zoom-in of imprinteddomains listing pilot WT reads. The Ezh2−/− library is depleted of thesereads. Freq≥3 reads shown. *, ncRNA.

FIG. 1K shows a pair of graphs. On the left is a graph showingalignments of distinct WT pilot reads to the mouse chromosome 12. Thenumber of reads per 100-kb window for both unique and repetitiveelements are plotted from centromere (CEN) to distal telomere (TELO).100-kilobase windows are nonoverlapping and consecutive. Reads werenormalized such that those mapping to ‘n’ locations were counted as1/nth of a read at each location. Chr, chromosome. Dark grey, forwardstrand; light grey, reverse strand. On the right is a graph showing azoom-in of imprinted domains listing pilot WT reads. The Ezh2−/− libraryis depleted of these reads. Freq≥3 reads shown. *, ncRNA.

FIG. 1L is a table showing the number of reads (with freq≥3) in eachgenetic category. Parentheses show the percent of distinct readsbelonging to each category (e.g., 50.7% of reads map to ncRNA).

FIG. 1M is a table showing the number of transcription units hit byreads with freq≥3. The total number transcripts in each category isindicated in column 2. Parentheses show the percent representation ofeach category in the PRC2 transcriptome (e.g., 13.7% of known Suz12domains are represented in the PRC2 transcriptome).

FIGS. 2A-2D show data related to larger-scale sequencing to capture thePRC2 transcriptome.

FIG. 2A is a scatterplot map showing 39,003 transcripts from the UCSCjoined transcriptome database by their RPKM values in the wildtypelibrary (x-axis) and the null library (y-axis). A UCSC transcript thatis neither represented in the WT or null library is plotted at (0,0).Smoothing was performed by the function, smoothScatter, in R. Darkershades correspond to a greater density of genes at a given point on thegraph. The 3:1 WT/null enrichment line and the x=0.4 threshold are shownas dotted grey lines. Transcripts meeting the criteria of ≥3:1 RPKMenrichment and WT RPKM≥0.4 are deemed strong positives and are shown inred, in a pool marked “PRC2 transcriptome”. Transcripts that fall belowthe cut-offs are considered background and are shown in orange. Tsix isoff-chart (arrow) with (x,y) coordinates indicated.

FIG. 2B is a table showing characteristics of the PRC2 transcriptome.Numbers in parentheses indicate the total number of genes in eachcategory (e.g., Of 793 tumor suppressors, 325 are found in the PRC2transcriptome).

FIG. 2C is a graph showing the results of higher resolution analysis ofthe X-inactivation center. Distinct reads were smoothed with a sliding200-bp window on the x-axis and their representations plotted on they-axis.

FIG. 2D is a graph showing the results of metagene analysis; distinctreads from the PRC2 transcriptome are plotted as a function of distancefrom TSS.

FIG. 2E is a pair of graphs showing the frequency of all reads plottedas a function of distance from the TSS for WT (left) and Ezh2−/− (right)libraries. The x-axis shows the promoter region of all genes taken fromthe UCSC refGene database, with coordinates in base-pairs (bp) relativeto the TSS. Forward strand reads are indicated in blue, reverse strandin red. Arrows indicate enrichment near TSS.

FIG. 2F is a set of three graphs showing the distinct reads (duplicatesremoved) plotted as a function of distance from the TSS for the WT,Ezh2−/−, and IgG samples. Arrows indicate enrichment near TSS.

FIGS. 3A-B are read density plots for Nesp/Gnas (A) and Dlk1/Gtl2 (B)imprinted clusters. Distinct reads are smoothed with sliding consecutive200-bp or 2-kb windows on the x-axis and their representations plottedon the y-axis. *, ncRNA. Chr, chromosome. Dark grey, forward strand;light grey, reverse strand. The Ezh2−/− library is depleted of thesereads.

FIGS. 4A-E: Confirmation by native RIP/qRT-PCR and UV-crosslinked RIP.

FIG. 4A is a set of nine bar graphs showing the results of qRT-PCR tocompare a-Ezh2 and IgG pulldowns. The experiments were performed 2-3times in triplicate. Error bar=1 standard deviation (SD). P wascalculated using the two-tailed student t-test. Asterisks, undetectablelevels.

FIG. 4B is a set of eight bar graphs showing the results of qRT-PCRafter native a-Ezh2 RIP of wildtype and null ES cells, each normalizedto IgG RIP values. Values for Xist, Gtl2-as, and Foxn2-as wereoff-chart. Experiments were performed 2-4 times in triplicate. 1 SDshown. P is calculated using the paired, two-tailed student t-test.Asterisks, undetectable RNA levels.

FIG. 4C is a set of seven bar graphs showing the results of Confirmationof native RIP by UV-crosslinked RIP. Each experiment was performed 2-4times in triplicate, normalized to IgG pulldowns, and compared to thatof Ezh2−/− controls using the t-test (P). 1 SD shown.

FIG. 4D is an image showing the results of Northern blot analysis ofindicated RNA species.

FIG. 4E is an image showing the results of Native RIP with RNAsepretreatment, followed by qRT-PCR quantification.

FIGS. 5A-F show the results of biochemical analysis demonstating directinteractions between RNA and PRC2.

FIG. 5A is an image showing a Coomassie-stained gel of human PRC2 andsubunits. Different migrations reflect Flag-tagged versus untaggedversions of each protein.

FIG. 5B is a schematic showing WT and mutant (Mut) versions of RepA (SEQID NO:193118 and 193119) and Hes1 (SEQ ID NO:193120 and 193121) asdouble stem-loop structures.

FIG. 5C is an image showing the results of RNA EMSA using purified PRC2complex and end-labeled probes. Negative controls: DsI and DsII, RNAsequences from Xist outside of RepA. Double shifts indicate presence ofmultiple subcomplexes of PRC2.

FIG. 5D is an image showing the results of RNA EMSA using purified PRC2subunits. The lanes were run on the same gel but separated in the imagebecause a lane was cut out between each panel.

FIG. 5E is an image showing the results of Titration of 1-25 fmoles ofHes1-as RNA probe against 0.1-1.0 mg of EZH2.

FIG. 5F is a set of four images showing the results of RNA pulldownassays using purified PRC2 and indicated RNA probes loaded in equalmoles. 25% of the IP fraction, 10% of flow-through, and 10% of RNA inputare shown.

FIGS. 6A-E show that Gtl2 controls Dlk1 by targeting PRC2.

FIG. 6A is a map of Dlk1-Gtl2 and the positions of shRNAs and primerpairs used in RIP and ChIP. Dotted lines indicate that the transcriptsmay extend further.

FIG. 6B is a set of three bar graphs showing qRT-PCR of Gtl2, Dlk1, andGtl2-as RNA levels after Gtl2 knockdown (KD) (left bar in each graph) orscrambled KD (right bar). Pools of knockdown cells are used. RNA levelsare normalized to Gapdh levels and compared to levels in scrambledknockdown controls (Scr). Experiments were performed in triplicates twotimes. One SD shown. P is calculated using a two-tailed student t-testbetween Gtl2 versus Scr KDs.

FIG. 6C is a pair of bar graphs showing qChIP of PRC2 association in KDcells. ChIP was carried out with α-Ezh2 (top) and a-H3K27me3 (bottom)antibodies, with normal rabbit IgG as control. qPCR levels are expressedas a percentage of input DNA. DMR, differentially methylated region.ICR, imprint control region. One SD shown. P, determined by two-tailedstudent t-tests of Gtl2 versus Scr KD.

FIG. 6D is a bar graph showing qRT-PCR of Ezh2 mRNA levels in Gtl2- andScr-KD clones.

FIG. 6E is a bar graph showing qRT-PCR of Dlk1 expression in Ezh2−/−versus WT cells relative to Gtl2 expression. One is a bar graph showingSD shown.

FIGS. 7A-C show RIP-seq and bioinformatics analysis in the test andcontrol samples.

FIG. 7A is a flow chart describing the RIP-seq and bioinformaticsanalysis in test and control samples.

FIG. 7B is an image showing that treating RIP products with RNAseA (10ug/mL) and RNAseV1 (0.001 U/mL) destroyed products in the 200-2,000 ntsize range (boxed), suggesting that the material pulled down was RNA.Bands in the <200 nt range are PCR primer dimers.

FIG. 7C is a set of three graphs showing the results of metageneanalysis: The number of distinct reads plotted as a function of distancefrom the TSS for the WT, Ezh2−/−, and IgG pilot samples (plotted to thesame scale).

FIGS. 8A-C show chromosome ideograms for the wildtype transcriptomeplotted with duplicates removed. Shown are alignments of all pilot readsafter removing duplicates in the wildtype transcriptome to the mousegenome. The number of reads per 100-kb window for both unique andrepetitive elements are plotted as a function of distance (in bp) fromcentromere (CEN) to distal telomere (TELO). 100-kilobase windows arenonoverlapping and consecutive. Reads were normalized such that thosemapping to ‘n’ locations were counted as 1/n^(th) of a read at eachlocation, and further normalized to account for the greatly reducedcomplexity of the control libraries relative to the IP samples. Chr,chromosome. Dark grey, forward strand; light grey, reverse strand.

FIGS. 9A-C show chromosome ideograms for the Ezh2−/− control libraryplotted with duplicates removed. The analysis was carried out asdescribed in FIG. 8 legend. Note that the graphs are plotted at scalesidentical to those for the wildtype library.

FIGS. 10A-C show chromosome ideograms for the IgG control libraryplotted with duplicates removed. The analysis was carried out asdescribed in FIG. 8 legend. Note that the graphs are plotted at scalesidentical to those for the wildtype library.

FIGS. 11A-C show chromosome ideograms for wildtype technical replicateplotted with duplicates removed. The analysis was carried out asdescribed in FIG. 8 legend. Note that the graphs are plotted at scalesidentical to those for the original wildtype library.

FIGS. 12A-C show chromosome ideograms for the wildtype biologicalreplicate plotted with duplicates removed. The analysis was carried outas described in FIG. 8 legend. Note that the graphs are plotted atscales identical to those for the original wildtype library.

FIG. 13 depicts a plot showing the region around the c-Myc oncogene(bar).

FIG. 14 depicts a plot showing the region around the Nkx2-1 gene (alsoknown as Titf1).

FIGS. 15A-C show that LNA molecules Targeting Xist Repeat C abolish XistRNA localization to Xi.

FIG. 15A is an alignment of 14 tandem mouse Repeat C (SEQ ID NOs:193122-193135). Conserved nucleotides are indicated with an asterisk.The regions targeted by LNA molecules are indicated by a line.

FIG. 15B is a pair of graphs showing quantitation of the results of XistRNA FISH at indicated timepoints after LNA molecule nucleofection.Results shown for LNA-C1, but LNA-C2 gives similar results.

FIG. 15C shows an alignment of mouse and human Repeat C regions targetedby the LNA molecules (left) (SEQ ID NOs: 193136-193139).

FIG. 16 shows Xist RNA displacement is accompanied by loss of PRC2localization and recovery occurs initially near Xist. The bar graphshows the results of real-time qRT-PCR analysis of Xist levels,normalized to Gapdh RNA.

FIGS. 17A-C show that a broader domain around Repeat C is required forXist localization.

FIG. 17A is a schematic map of Xist exon/intron structure and locationsof LNA molecules utilized.

FIG. 17B is a bar graph showing the results of qRT-PCR of Xist levels,normalized to Gapdh quantities.

FIG. 17C is an image of a Western blot with Ezh2 antibodies. Actin isused as a loading control.

FIGS. 18A-D show Ezh2 recovery after LNA molecule nucleofection isuniform along Xi but slow in kinetics.

FIG. 18A is a schematic representation of X-genes.

FIG. 18B is a bar graph showing Ezh2 ChIP analysis at X-genes.

FIG. 18C is a pair of bar graphs showing ChIP analysis of Ezh2 after LNAmolecule nucleofection. Asterisks, P<0.05 by the Student t-Test.

FIG. 18D is a bar graph showing ChIP analysis of Ezh2 enrichment on theautosomal En1 promoter.

Table 1: Imprinted regions hit by the PRC2 transcriptome. As usedherein, Table 1 refers to Table 1 of International Patent ApplicationNo. PCT/US2011/060493, published as WO 2012/065143, which isincorporated herein by reference in its entirety.

Intersection of the PRC2 transcriptome with imprinted gene coordinates(available online at geneimprint.com). The murine imprinted gene (i.e.,an intersecting or nearby gene) targeted by the PRC2-binding transcriptis shown in column 1. Column 1 also shows the chromosome strand of themurine imprinted gene (“+”sign indicates that the gene is transcribedfrom the top or plus strand, while “−” sign indicates that thePRC2-binding transcript is transcribed from the bottom or minus strand).The chromosome localization and nucleotide coordinates in mm9 of thePRC2-binding transcript are shown in column 2, as well as a “+”sign or“−” sign that indicates whether the PRC2-binding transcript istranscribed from the top strand (plus strand hit) or bottom strand(minus strand hit). Column 3 displays the SEQ ID NO: of the mousePRC2-binding transcript (i.e., the nucleotide sequence transcribed fromthe mouse chromosomal coordinates and strand of column 2, converted toRNA by replacing T with U). Column 4 shows the corresponding human genename for the murine imprinted gene of column 1, obtained from the MouseGenome Database (MGD), Mouse Genome Informatics, The Jackson Laboratory,Bar Harbor, Me. World Wide Web (www.informatics.jax.org). Mouse-to-humanLiftOver of the mouse chromosome coordinates in column 2, performed inthe UCSC genome browser as described herein, generated the orthologoushuman chromosome coordinates which appear in Column 5. 50% conservationwas used for LiftOver analysis. Additional human chromosome coordinateswere generated by mapping of highly conserved or homologous regions fromthe mouse to human genome. Column 6 displays the SEQ ID NO: of thepredicted human PRC2-binding transcript (i.e., the nucleotide sequencetranscribed from the human chromosomal coordinates and strand of column5, converted to RNA by replacing T with U). When the PRC2-interactingtranscript is transcribed from the opposite strand compared to theimprinted reference gene in column 1, that implies that thePRC2-interacting RNA is complementary, or antisense-strand (“oppositestrand”) in orientation, to the reference imprinted gene. Note that thePRC2-binding transcript need not be the reference imprinted gene itself,but a distinct transcript that overlaps in position.

Table 2: The PRC2 transcriptome. As used herein, Table 2 refers to Table2 of International Patent Application No. PCT/US2011/060493, publishedas WO 2012/065143, which is incorporated herein by reference in itsentirety.

Shown are the 9,788 transcripts associated with PRC2 in mouse ES cells.The joined UCSC transcriptome was used to map reads in the WT library.UCSC transcripts hit by reads are shown in column 1 (“MTR” joined genename). Column 2 shows the chromosome strand of the UCSC transcript(“+”sign indicates that the top or plus strand, while “−” sign indicatesthe bottom or minus strand). The chromosome localization and nucleotidecoordinates in mm9 of the PRC2-binding transcript are shown in column 3,as well as a “+”sign or “−” sign that indicates whether the PRC2-bindingtranscript is transcribed from the top strand (plus strand hit) orbottom strand (minus strand hit). RPKM values of 0.4 or above wereconsidered a “hit”. Column 4 displays the SEQ ID NO: of the mousePRC2-binding transcript (i.e., the nucleotide sequence transcribed fromthe mouse chromosomal coordinates and strand of column 3, converted toRNA by replacing T with U). Mouse-to-human LiftOver of the mousechromosome coordinates in column 3, performed in the UCSC genome browseras described herein, generated the orthologous human chromosomecoordinates which appear in column 5. Additional human chromosomecoordinates were generated by mapping of highly conserved or homologousregions from the mouse to human genome. Column 6 displays the SEQ ID NO:of the predicted human PRC2-binding transcript (i.e., the nucleotidesequence transcribed from the human chromosomal coordinates and strandof column 5, converted to RNA by replacing T with U). 50% conservationwas used for LiftOver analysis. Alignment of reads are reported based onthe chromosomal strand the reads match, regardless of the orientation ofthe overlapping gene. A single hit to the opposite strand of thereference transcript implies that the PRC2-binding RNA is complementary,or antisense-strand (“opposite strand”) in orientation, to the referencetranscript. Any overlapping refGene targeted by the murine PRC2-bindingtranscript of column 2 (i.e., an intersecting or nearby gene), withoutregard to orientation, is shown in column 7.

Table 3: Bivalent domains with an associated PRC2-interactingtranscript. As used herein, Table 3 refers to Table 3 of InternationalPatent Application No. PCT/US2011/060493, published as WO 2012/065143,which is incorporated herein by reference in its entirety.

Intersection of the PRC2 transcriptome with ES-cell bivalent domains.mm8 coordinates from Mikkelsen et al. (2007) were converted to mm9 forthis analysis. Column 1 displays the overlapping refGenes (i.e., anintersecting or nearby gene) targeted by the PRC2-binding transcript ofcolumn 2, regardless of strand orientation. Chromosome coordinates (mm9)of the PRC2-binding transcript are shown in column 2, as well as a“+”sign or “−” sign that indicates whether the PRC2-binding transcriptis transcribed from the top (plus) or bottom (minus) strand. Column 3displays the SEQ ID NO: of the mouse PRC2-binding RNA (i.e., thenucleotide sequence transcribed from the mouse chromosomal coordinatesand strand of column 2, converted to RNA by replacing T with U).Mouse-to-human LiftOver of the mouse chromosome coordinates in column 2(50% conservation was used for LiftOver analysis), performed in the UCSCgenome browser as described herein, generated the orthologous humanchromosome coordinates which appear in column 4. Additional humanchromosome coordinates were generated by mapping of highly conserved orhomologous regions from the mouse to human genome. Column 5 displays theSEQ ID NO: of the predicted human PRC2-binding RNA (i.e., the nucleotidesequence transcribed from the human chromosomal coordinates and strandof column 3, converted to RNA by replacing T with U).

Table 4: PRC2-binding sites with an associated PRC2-interactingtranscript. As used herein, Table 4 refers to Table 4 of InternationalPatent Application No. PCT/US2011/060493, published as WO 2012/065143,which is incorporated herein by reference in its entirety.

Intersection of the PRC2 transcriptome with known Suz12-binding sites inES cells. mm8 coordinates from Boyer et al., 2006, were converted tomm9. Column 1 displays the overlapping refGenes (i.e., an intersectingor nearby gene) targeted by the murine PRC2-binding transcript of column2, regardless of strand orientation. Chromosome coordinates of thePRC2-binding transcript are shown in column 2, as well as a “+”sign or“−” sign that indicates whether the PRC2-binding transcript istranscribed from the top (plus) or bottom (minus) strand. Column 3displays the SEQ ID NO: of the mouse PRC2-binding transcript (i.e., thenucleotide sequence transcribed from the mouse chromosomal coordinatesand strand of column 2, converted to RNA by replacing T with U).Mouse-to-human LiftOver of the mouse chromosome coordinates in column 2(50% conservation was used for LiftOver analysis), performed in the UCSCgenome browser as described herein, generated the orthologous humanchromosome coordinates which appear in column 4. Additional humanchromosome coordinates were generated by mapping of highly conserved orhomologous regions from the mouse to human genome. Column 5 displays theSEQ ID NO: of the predicted corresponding human PRC2-binding transcript(i.e., the nucleotide sequence transcribed from the human chromosomalcoordinates and strand of column 4, converted to RNA by replacing T withU).

Table 5: LincRNA domains intersecting with the PRC2 transcriptome. Asused herein, Table 5 refers to Table 5 of International PatentApplication No. PCT/US2011/060493, published as WO 2012/065143, which isincorporated herein by reference in its entirety.

Intersection of the PRC2 transcriptome with mouse lincRNA domains(Guttman et al., 2009). Coordinates were converted from mm6 to mm8 andthen to mm9, as there is no direct LiftOver from mm6 to mm9. Hits canoccur to either strand of the lincRNA domains. Chromosome coordinates ofthe PRC2-binding transcript are shown in column 1, as well as a “+”signor “-” sign that indicates whether the PRC2-binding transcript istranscribed from the top (plus) or bottom (minus) strand. Column 2displays the SEQ ID NO: of the mouse PRC2-binding RNA (i.e., thenucleotide sequence transcribed from the mouse chromosomal coordinatesand strand of column 1, converted to RNA by replacing

T with U). Mouse-to-human LiftOver of the mouse chromosome coordinatesin column 1 (50% conservation was used for LiftOver analysis), performedin the UCSC genome browser as described herein, generated theorthologous human chromosome coordinates which appear in column 3.Additional human chromosome coordinates were generated by mapping ofhighly conserved or homologous regions from the mouse to human genome.Column 4 displays the SEQ ID NO: of the predicted corresponding humanPRC2-binding transcript (i.e., the nucleotide sequence transcribed fromthe human chromosomal coordinates and strand of column 3, converted toRNA by replacing T with U). Overlapping refGenes targeted by the murinePRC2-binding transcript of column 1 (i.e., an intersecting or nearbygene) regardless of strand orientation, are shown in column 5.

Table 6: Hits to the PRC2 transcriptome within oncogene loci. As usedherein, Table 6 refers to Table 6 of International Patent ApplicationNo. PCT/US2011/060493, published as WO 2012/065143, which isincorporated herein by reference in its entirety.

Intersection of the PRC2 transcriptome with known oncogene loci(available online at cbio.mskcc.org/CancerGenes). Human oncogene lociwere mapped to mouse coordinates for this analysis by first mergingcoordinates on the same strand and chromosome of same named genes in therefGene, then intersecting identical names of the oncogenes with that ofthe genes in refGene without regard to capitalization. Column 1 showsthe mouse gene name corresponding to the human oncogene of column 6targeted by the PRC2-binding transcript. Corresponding mouse and humangene names were obtained from the Mouse Genome Database (MGD), MouseGenome Informatics, The Jackson Laboratory, Bar Harbor, Maine(www.informatics.jax.org). Column 1 also shows the chromosome strand ofthe mouse oncogene (“+”sign indicates that the gene is transcribed fromthe top or plus strand, while “−” sign indicates that the gene istranscribed from the bottom or minus strand). The chromosomelocalization and nucleotide coordinates in mm9 of the PRC2-bindingtranscript are shown in column 2, as well as a “+”sign or “−” sign thatindicates whether the PRC2-binding transcript is transcribed from thetop (plus) or bottom (minus) strand. Column 3 displays the SEQ ID NO: ofthe mouse PRC2-binding transcript (i.e., the nucleotide sequencetranscribed from the mouse chromosomal coordinates and strand of column2, converted to RNA by replacing T with U). Mouse-to-human LiftOver ofthe mouse chromosome coordinates in column 2 (50% conservation was usedfor LiftOver analysis), performed in the UCSC genome browser asdescribed herein, generated the orthologous human chromosome coordinateswhich appear in column 4. Additional human chromosome coordinates weregenerated by mapping of highly conserved or homologous regions from themouse to human genome. Column 5 displays the SEQ ID NO: of the predictedcorresponding human PRC2-binding transcript (i.e., the nucleotidesequence transcribed from the human chromosomal coordinates and strandof column 4, converted to RNA by replacing T with U). Column 6 shows hehuman refGene names of each murine oncogene (i.e., an intersecting ornearby gene) targeted by the murine PRC2-binding transcript of column 2.When the PRC2-binding transcript is on the opposite strand from therefGene, it implies that the PRC2-interacting RNA is complementary, oranti sense-strand (“opposite strand”) in orientation to the referenceoncogene. Note that the PRC2-interacting transcript need not be therefGene itself, but a distinct transcript that overlaps in position withthe refGene. The cancers associated with the oncogene are shown incolumn 7.

Table 7: Hits to the PRC2 transcriptome within tumor suppressor loci. Asused herein, Table 7 refers to Table 7 of International PatentApplication No. PCT/US2011/060493, published as WO 2012/065143, which isincorporated herein by reference in its entirety.

Intersection of the PRC2 transcriptome with known tumor suppressor loci(available online at cbio.mskcc.org/CancerGenes). Human tumor suppressorloci were mapped to mouse coordinates for this analysis in a similarmanner to the oncogenes of Table 6. The table is organized as describedin Table 6 legend. Column 1 shows the mouse tumor suppressorcorresponding to the human tumor suppressor of column 6 targeted by thePRC2-binding transcript. Corresponding mouse and human gene names wereobtained from the Mouse Genome Database (MGD), Mouse Genome Informatics,The Jackson Laboratory, Bar Harbor, Me. (www.informatics.jax.org).Column 1 also shows the chromosome strand of the mouse tumor suppressor(“+”sign indicates that the gene is transcribed from the top or plusstrand, while “-” sign indicates that the gene is transcribed from thebottom or minus strand). The chromosome localization and nucleotidecoordinates in mm9 of the PRC2-binding transcript are shown in column 2,as well as a “+”sign or “-” sign that indicates whether the PRC2-bindingtranscript is transcribed from the top (plus) or bottom (minus) strand.Column 3 displays the SEQ ID NO: of the mouse PRC2-binding RNA (i.e.,the nucleotide sequence transcribed from the mouse chromosomalcoordinates and strand of column 2, converted to RNA by replacing T withU). Mouse-to-human LiftOver of the mouse chromosome coordinates incolumn 2 (50% conservation was used for LiftOver analysis), performed inthe UCSC genome browser as described herein, generated the orthologoushuman chromosome coordinates which appear in column 4. Additional humanchromosome coordinates were generated by mapping of highly conserved orhomologous regions from the mouse to human genome. Column 5 displays theSEQ ID NO: of the predicted corresponding human PRC2-binding transcript(i.e., the nucleotide sequence transcribed from the human chromosomalcoordinates and strand of column 4, converted to RNA by replacing T withU). Column 6 shows the human refGene names of each tumor suppressor(i.e., an intersecting or nearby gene) targeted by the murinePRC2-binding transcript of column 2.

Table 8: Intersection of the PRC2 transcriptome, and Peaks generated byoverlapping reads in Appendix I, with target genes. As used herein,Table 8 refers to Table 8 of International Patent Application No.PCT/US2011/060493, published as WO 2012/065143, which is incorporatedherein by reference in its entirety.

The sequence reads in Appendix I (obtained from sequencing cDNAaccording to Examples 1-2) represent regions protected from endogenousnucleases during the RIP procedure and thus represent regions of RNAthat bind to PRC2. As noted above, Appendix I appears in U.S. Prov.Appin. No. 61/425,174 filed on Dec. 20, 2010, which is not attachedhereto but is incorporated by reference herein in its entirety, Thesesequence reads of Appendix I that were enriched 3:1 in WT vs. null andshowed a minimal RPKM value of 0.4 were overlapped to generate longercontiguous regions of sequence referred to herein as a “Peak.” Thecorresponding nucleotide sequences of the mouse Peaks (converted to RNAby replacing T with U) appear in the sequence listing as SEQ ID NOS:21583 to 124436, or 190717 to 190933, or 191088. Mouse-to-human LiftOverof the mouse chromosome coordinates and strand of these mouse Peaks wasperformed in the UCSC genome browser as described herein, to generateorthologous human chromosome coordinates. Each corresponding human PeakRNA sequence (i.e., the nucleotide sequence of the human chromosomalcoordinates and strand, converted to RNA by replacing T with U) appearin the sequence listing as SEQ ID NOS: 124437 to 190716, or 190934 to191086, or 191087.

These human Peaks and the human PRC2 transcriptome (i.e. human sequencesof

PRC2-binding transcripts referenced in Tables 1-7) were intersected withknown genes from the NCBI refGene database to identify genes targeted bythe PRC2-binding RNA (i.e. an intersecting or nearby gene). Similarly,the mouse Peaks and the mouse PRC2 transcriptome of Tables 1-7 wereintersected with known genes from the NCBI refGene database to identifygenes targeted by the PRC2-binding RNA (i.e. an intersecting or nearbygene).

Columns 1 and 2 displays the SEQ ID NO: of the sequence of all of (a)the human PRC2-binding transcripts, (b) the human Peak sequences withinthe PRC2-binding RNA, (c) the mouse PRC2-binding transcripts, and (d)the mouse Peak sequences within the PRC2-binding RNA, which target theNCBI gene (i.e., are intersecting or nearby) shown in Column 3. Column 3shows the NCBI gene name and unique NCBI gene ID number (NationalLibrary of Medicine (US), National Center for Biotechnology Information;chapter 19, Entrez Gene: A Directory of Genes.www.ncbi.nlm.nih.gov/gene/). Human gene names appear as all capitals,while mouse gene names appear with only the first letter capitalized.

Column 1 displays SEQ ID NOs for “same strand” PRC2-binding RNA that istranscribed from the same strand as the reference NCBI gene (forexample, if the NCBI gene is transcribed from the minus strand of thechromosome, then the PRC2-binding RNA is also transcribed from the minusstrand). Column 2 displays SEQ ID NOs for “opposite strand” PRC2-bindingRNA that is transcribed from the opposite strand, or antisense-strand,compared to the reference NCBI gene. SEQ ID NOs. from 1 to 21582 or191089 to 192972 represent transcripts, while SEQ ID NOs. from 21583 to191088 represent Peaks. In columns 1 and 2, the degree of overlapbetween (a) the transcript or Peak coordinates and (b) the NCBI genecoordinates appears in square brackets.A positive number indicates thenumber of overlapping nucleotides between the two, and a negative numberrepresents the size of the gap between the two (i.e. the number ofnucleotides of distance between the two). For Peaks, an “F” within thesquare brackets indicates that the Peak coordinates fully overlap thegene coordinates. For transcripts, an “F” within the square bracketsindicates that the transcript coordinates fully overlap the genecoordinates, or vice versa.

Table 9: Categories of PRC2-binding RNA, genes targeted by the RNA, anduses in treatment of disease. As used herein, Table 9 refers to Table 9of International Patent Application No. PCT/US2011/060493, published asWO 2012/065143, which is incorporated herein by reference in itsentirety.

Column 1 shows the NCBI gene name and unique gene ID. Column 2 are thecategories of functional groups of genes, and the diseases, disorders orconditions that are associated with these genes and can be treated bymodulating their expression. Column 3 is the description of the genefrom NCBI.

APPENDIX I of U.S. provisional application 61/425,174 filed on Dec. 20,2010, the entirety of which is incorporated by reference herein, is alisting of the complete RIP-seq dataset, showing all of the reads in thedataset. Appendix I is not attached hereto. The sequence reads inAppendix I come directly off the Illumina GA-II genome analyzer and arein an orientation that is the reverse complement of the PRC2-bindingtranscript. Appendix I is a filtered subset of all of the reads afterbioinformatic filtering removed adaptor/primer dimers, mitochondrialRNA, rRNA, homopolymers, reads with indeterminate nucleotides, andtruncated reads (<15nt).

DETAILED DESCRIPTION

The RIP-seq technology described herein was used to capture agenome-wide pool of long transcripts (>200 nt) that bind with the PRC2complex, directly or indirectly. The PRC2 transcriptome described hereinconsists of ˜10,000 RNAs in mouse ES cells, likely accounting for 5-25%of expressed sequences in mice, depending on the actual size of thetotal mouse transcriptome. Transcriptome characterization has identifiedclasses of medically significant targets, including dozens of imprintedloci, hundreds of oncogene and tumor suppressor loci, and multiplestem-cell-related domains, some of which may be used as biomarkers andtherapeutics targets in the future. Many if not all of the mousePRC2-transcripts have direct counterparts in the human epigenome.

As demonstrated herein, at least a subset of RNAs directly interactswith Polycomb proteins in vivo and, in many cases, the interactingsubunit is Ezh2. A recent study indicates that Suz12 also interacts withRNA (Kanhere et al., 2010). Differences between bacterially- andbaculovirus-produced subunits could result in varying post-translationalmodifications with effects on binding properties. However, it is likelythat multiple subunits of PRC2 can be regulated by RNA (especially Ezh2and Suz12, both of which have nucleic-acid binding motifs), which couldmodulate binding between PRC2 subunits, binding affinities of PRC2 forchromatin, and/or Ezh2 catalytic rates. This scenario would amplify thenumber of potential mechanisms by which RNA regulates Polycomb. Thepresent study suggests thousands of RNA cofactors for Ezh2, the baitused for RIP-seq, specifically as part of the PRC2 complex. To thepresent inventors' knowledge, Ezh2 is only present in Polycombcomplexes, as biochemical purification using tagged Ezh2 identifies onlyPolycomb-related peptides (Li et al., 2010) and knocking out othersubunits of PRC2 results in rapid degradation of Ezh2 (Pasini et al.,2004; Montgomery et al., 2005; Schoeftner et al., 2006).

Both cis and trans mechanisms may be utilized by RNAs in the PRC2transcriptome. While it has been postulated that HOTAIR works in trans(Rinn et al., 2007; Gupta et al.), the large number of antisensetranscripts in the transcriptome suggests that many, like Tsix, mayfunction by directing PRC2 to overlapping or linked coding loci in cis.Provided herein is the example of a linked RNA, Gtl2, which binds andtargets PRC2 to Dlk1 locus to direct H3K27 trimethylation in cis.

The evidence presented herein demonstrates that RNA cofactors are ageneral feature of Polycomb regulation and that inhibitory nucleic acidsas described herein that target RNA in the PRC2 transcriptome cansuccessfully up-regulate gene expression, presumably by inhibitingPRC2-associated repression. Genes in cis, in either antisense-strandorientation or same strand orientation, and extending 1 kb or more fromthe location of the PRC2-binding RNA, can be regulated. Regulation byRNA need not be specific to Polycomb proteins. RIP-seq technology can beutilized to identify RNA cofactors for other chromatin modifiers, anddifferent cell types might have distinct transcriptomes consistent withtheir developmental profiles. Because chromatin modifiers such as PRC2play a central role in maintaining stem cell pluripotency and in cancer,a genome-wide profile of regulatory RNAs will be a valuable resource inthe quest to diagnose and treat disease.

RIP-Seq-Methods of Producing Long Non-Coding RNAs

Described herein are methods for producing libraries of lncRNAs. Thesemethods were used to identify RNAs that bind the Ezh2 portion of thePRC2 complex, but does not exclude contacts with other PRC2 subunits orassociated proteins. In some embodiments, the methods include the stepsshown in FIG. 1A; one of skill in the art will appreciate that othertechniques can be substituted for those shown.

In some embodiments, the methods include providing a sample comprisingnuclear ribonucleic acids (“nRNAs”), e.g., a sample comprising nuclearlysate, e.g., comprising nRNAs bound to nuclear proteins; contacting thesample with an agent, e.g., an antibody, that binds specifically to anuclear protein that is known or suspected to bind to nuclearribonucleic acids. Some examples of nuclear proteins that are known orsuspected to bind to nuclear ribonucleic acids include Ezh2 (Zhao etal., Science. 2008 Oct. 31; 322(5902):750-6; Khalil et al., Proc NatlAcad Sci USA. 2009 Jul. 14; 106(28):11667-72. Epub 2009 Jul. 1); G9a(Nagano et al., Science. 2008 Dec. 12; 322(5908):1717-20. Epub 2008 Nov6); and Cbx7 (Yap et al., Mol Cell. 2010 Jun. 11; 38(5):662-74.)

In some embodiments, the methods are applied under conditions sufficientto form complexes between the agent and the protein, and include some orall of the following: isolating the complexes; synthesizing DNAcomplementary to the nRNAs to provide an initial population of cDNAs;PCR-amplifying, if necessary, using strand-specific primers; purifyingthe initial population of cDNAs to obtain a purified population of cDNAsthat are at least 20 nucleotides (nt) in length; and high-throughputsequencing the purified population of cDNAs. Homopolymer reads arefiltered, and reads matching the mitochondrial genome and ribosomal RNAsare excluded from all subsequent analyses. Reads that align to areference genome with ≤1 mismatch are retained, excluding homopolymers,reads that align to the mitochondrial genome, and ribosomal RNAs. Highprobability PRC2-interacting transcripts are then called based on twocriteria: (1) that the candidate transcript has a minimum read densityin RPKM terms (number of reads per kilobase per million reads); and/or(2) that the candidate transcript is enriched in the wildtype libraryversus a suitable control library (such as a protein-null library orlibrary made from an IgG pulldown done in parallel).

In general, to construct RIP-seq libraries, cell nuclei are prepared,treated with DNAse, and incubated with antibodies directed against achromatin-associated factor of interest, along with a control IgGreaction in parallel. RNA-protein complexes are then immunoprecipitatedwith agarose beads, magnetic beads, or any other platform in solution oron a solid matrix (e.g., columns, microfluidic devices). RNAs areextracted using standard techniques. To capture all RNAs (not just polyARNAs) and to preserve strand information, asymmetric primers are used togenerate cDNA from the RNA template, in which the first adaptor(adaptorl) to make the first strand cDNA contains a random multimersequence (such as random hexamers) at the 3′ end. A reversetranscriptase is used to create the first strand. A distinct secondadaptor (adaptor2) is used to create the second strand. One example isas follows: If Superscript II is used, it will add non-template CCC 3′overhangs, which can then be used to hybridize to a second adaptorcontaining GGG at the 3′ end, which anneal to the non-template CCCoverhangs. Other methods of creating second strands may be substituted.PCR using adaptor1- and adaptor2-specific primer pairs is then theperformed to amplify the cDNAs and the products sequenced via standardmethods of high throughput sequencing. Prior to sequencing, asize-selection step can be incorporated (if desired) in which RNAs orcDNAs of desired sizes are excised after separation by gelelectrophoresis (e.g., on a Nu-Sieve agarose gel or in an acrylamidegel) or other methods of purification, such as in a microfluidic deviceor in standard biochemical columns.

lncRNAs and lncRNA Libraries

The present invention includes the individual lncRNAs described herein,as well as libraries of lncRNAs produced by methods described herein. Insome embodiments, the libraries are in solution, or are lyophilized. Insome embodiments, the libraries are bound to a substrate, e.g., whereineach member of the library is bound to an individually addressablemember, e.g., an individual area on an array (e.g., a microarray), or abead. The PRC2-interacting RNA transcript, although non-coding, mayinclude a protein-coding sequence of bases if it is a distincttranscript that overlaps in position with a protein-coding referencegene (e.g. the gene whose expression is modulated in cis).

In one embodiment, a lncRNA includes a nucleotide sequence that is atleast about 85% or more homologous or identical to the entire length ofa lncRNA sequence shown herein, e.g., in Table 2, 3, 4, or 5, or afragment comprising at least 20 nt thereof (e.g., at least 25, 30, 35,40, 50, 60, 70, 80, 90, or 100 nt thereof, e.g., at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 50% or more of the full length lncRNA). In someembodiments, the nucleotide sequence is at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous or identical to alncRNA sequence shown herein. In some embodiments, the nucleotidesequence is at least about 85%, e.g., is at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous or identical to alncRNA sequence described herein, in a fragment thereof or a region thatis much more conserved, such as Repeat A, but has lower sequenceidentity outside that region.

Mouse-to-human LiftOver analysis and analysis in the UCSC genome browserof syntenic positions indicate the existence of similar transcripts inthe human genome. This process and LiftOver chains are generallydescribed in Kent et al., Proc. Nat'l Acad. Sci., 100(20) 11484-11489(2003). Given the geographic and sequence similarities between the mouseand human transcripts, we believe that a similar number ofPRC2-interacting transcripts occur in the human system. For example, thePvt1 transcript described below is well conserved in humans. Human PVT1also occurs near the MYC gene and has a similar expression profile.Human PVT1 is frequently interrupted in Plasmacytomas and can also bedisrupted in Burkitt's lymphoma. Mouse Gtl2 and Xist are also wellconserved in the human system (GTL2/MEG3 and XIST). Thus, the datasuggest that many if not all of the mouse PRC2-transcripts have directcounterparts in the human epigenome. Such direct counterparts in otherspecies are termed “orthologous” herein.

LncRNAs may be functionally conserved without being highly conserved atthe level of overall nucleotide identity. For example, mouse Xist showsonly 76% overall nucleotide identity with human XIST using sliding 21-bpwindows, or an overall sequence identity of only 60%. However, withinspecific functional domains, such as Repeat A, the degree ofconservation can be >70% between different mammalian species. Thecrucial motif in Repeat A is the secondary structures formed by therepeat. A lncRNA interacting with PRC2 may therefore be similarly low inoverall conservation but still have conservation in secondary structurewithin specific domains of the RNA, and thereby demonstrate functionalconservation with respect to recruitment of PRC2.

Calculations of homology or sequence identity between sequences (theterms are used interchangeably herein) are performed as follows.

To determine the percent identity of two nucleic acid sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). The length of a reference sequencealigned for comparison purposes is at least 80% of the length of thereference sequence, and in some embodiments is at least 90% or 100%. Thenucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein nucleic acid “identity” is equivalent to nucleic acid“homology”). The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.

For purposes of the present invention, the comparison of sequences anddetermination of percent identity between two sequences can beaccomplished using a Blossum 62 scoring matrix with a gap penalty of 12,a gap extend penalty of 4, and a frameshift gap penalty of 5.

There are several potential uses for the lncRNAs described herein in thePRC2 transcriptome: The RNAs themselves, or antagomirs and smallmolecules designed against them, can be utilized to modulate expression(either up or down) of Polycomb target genes.

In various related aspects, including with respect to the targeting oflong ncRNAs by LNA molecule, long ncRNAs can include endogenous cellularRNAs that are greater than 60 nt in length, e.g., greater than 100 nt,e.g., greater than 200 nt, have no positive-strand open reading framesgreater than 100 amino acids in length, are identified as lncRNAs byexperimental evidence, and are distinct from known (smaller)functional-RNA classes (including but not limited to ribosomal,transfer, and small nuclear/nucleolar RNAs, siRNA, piRNA, and miRNA).See, e.g., Lipovich et al., “MacroRNA underdogs in a microRNA world:Evolutionary, regulatory, and biomedical significance of mammalian longnon-protein-coding RNA” Biochimica et Biophysica Acta (2010)doi:10.1016/j.bbagrm.2010.10.001; Ponting et al., Cell 136(4):629-641(2009), Jia et al., RNA 16 (8) (2010) 1478-1487, Dinger et al., NucleicAcids Res. 37 1685 (2009) D122-D126 (database issue); and referencescited therein. LncRNAs have also been referred to as long RNA, largeRNA, macro RNA, intergenic RNA, and NonCoding Transcripts.

The methods described herein can be used to target nuclear-localizedlncRNAs. Known classes of lncRNAs include large intergenic non-codingRNAs (lincRNAs, see, e.g., Guttman et al., Nature. 2009 Mar12;458(7235):223-7. Epub 2009 Feb. 1, which describes over a thousandexemplary highly conserved large non-coding RNAs in mammals; and Khalilet al., PNAS 106(28)11675-11680 (2009)); promoter associated short RNAs(PASRs; see, e.g., Seila et al., Science. 2008 Dec. 19;322(5909):1849-51. Epub 2008 Dec. 4; Kanhere et al., Molecular Cell 38,675-688, (2010)); endogenous antisense RNAs (see, e.g., Numata et al.,BMC Genomics. 10:392 (2009); Okada et al., Hum Mol Genet. 17(11):1631-40(2008); Numata et al., Gene 392(1-2):134-141 (2007); and Røsok andSioud, Nat Biotechnol. 22(1):104-8 (2004)); and RNAs that bind chromatinmodifiers such as PRC2 and LSD1 (see, e.g., Tsai et al., Science. 2010Aug 6;329(5992):689-93. Epub 2010 Jul. 8; and Zhao et al., Science. 2008Oct. 31; 322(5902):750-6).

Exemplary lncRNAs include XIST, TSIX, MALAT1, RNCR2, and HOTAIR. Thesequences for more than 17,000 long human ncRNAs can be found in theNCode™ Long ncRNA Database on the Invitrogen website. Additional longncRNAs can be identified using, e.g., manual published literature,Functional Annotation of Mouse (FANTOM3) project, Human Full-length cDNAAnnotation Invitational (H-Invitational) project, antisense ncRNAs fromcDNA and EST database for mouse and human using a computation pipeline(Zhang et al., Nucl. Acids Res. 35 (suppl 1): D156-D161 (2006); Engstromet al., PLoS Genet. 2:e47 (2006)), human snoRNAs and scaRNAs derivedfrom snoRNA-LBME-db, RNAz (Washietl et al. 2005), Noncoding RNA Search(Torarinsson, et al. 2006), and EvoFold (Pedersen et al. 2006).

Methods of Modulating Gene Expression

The lncRNAs described herein, including fragments thereof that are atleast 20 nt in length, and inhibitory nucleic acids and small moleculestargeting (e.g., complementary to) them, can be used to modulate geneexpression in a cell, e.g., a cancer cell, a stem cell, or other normalcell types for gene or epigenetic therapy. The cells can be in vitro,including ex vivo, or in vivo (e.g., in a subject who has cancer, e.g.,a tumor).

The methods described herein can be used for modulating expression ofoncogenes and tumor suppressors in cells, e.g., cancer cells. Forexample, to decrease expression of an oncogene in a cell, the methodsinclude introducing into the cell a long non-coding RNA, including aPRC2-binding fragment thereof, that regulates the oncogene as set forthin Table 6, imprinted genes in Table 1, and/or other growth-promotinggenes in Table 2.

As another example, to increase expression of a tumor suppressor in acell, the methods include introducing into the cell an inhibitorynucleic acid or small molecule that specifically binds, or iscomplementary, to a long non-coding RNA targeting a tumor suppressor asset forth in Table 7, imprinted genes in Table 1, and/or othergrowth-suppressing genes in Table 2 (e.g., Nkx2-1 or Titf-1, e.g., insubjects with cancer, e.g., lung adenocarcinoma patients). In someembodiments, the methods include introducing into the cell an inhibitorynucleic acid that specifically binds, or is complementary, to a longnon-coding RNA targeting an imprinted gene as set forth in Table 1. Anucleic acid that binds “specifically” binds primarily to the targetlncRNA or related lncRNAs to inhibit regulatory function of the lncRNAbut not of other non-target RNAs. The specificity of the nucleic acidinteraction thus refers to its function (e.g. inhibiting thePRC2-associated repression of gene expression) rather than itshybridization capacity. Inhibitory nucleic acids may exhibit nonspecificbinding to other sites in the genome or other mRNAs, without interferingwith binding of other regulatory proteins and without causingdegradation of the non-specifically-bound RNA. Thus this nonspecificbinding does not significantly affect function of other non-target RNAsand results in no significant adverse effects.

These methods can be used to treat cancer in a subject, by administeringto the subject a composition (e.g., as described herein) comprising anlncRNA (e.g., a lncRNA that inhibits a cancer-promoting oncogene orimprinted gene) or a PRC2-binding fragment thereof and/or an inhibitorynucleic acid that binds to a long non-coding RNA (e.g., an inhibitorynucleic acid that binds to a lncRNA that inhibits a tumor suppressor orcancer-suppressing imprinted gene and/or other growth-suppressing genesin Table 2).

Examples of genes involved in cancer and categories of cancer are shownin Table 9. Examples of cellular proliferative and/or differentiativedisorders include cancer, e.g., carcinoma, sarcoma, metastatic disordersor hematopoietic neoplastic disorders, e.g., leukemias. A metastatictumor can arise from a multitude of primary tumor types, including butnot limited to those of prostate, colon, lung, breast and liver origin.

As used herein, treating includes “prophylactic treatment” which meansreducing the incidence of or preventing (or reducing risk of) a sign orsymptom of a disease in a patient at risk for the disease, and“therapeutic treatment”, which means reducing signs or symptoms of adisease, reducing progression of a disease, reducing severity of adisease, in a patient diagnosed with the disease. With respect tocancer, treating includes inhibiting tumor cell proliferation,increasing tumor cell death or killing, inhibiting rate of tumor cellgrowth or metastasis, reducing size of tumors, reducing number oftumors, reducing number of metastases, increasing 1-year or 5-yearsurvival rate.

As used herein, the terms “cancer”, “hyperproliferative” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. “Pathologic hyperproliferative” cells occur in diseasestates characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair.

The terms “cancer” or “neoplasms” include malignancies of the variousorgan systems, such as affecting lung (e.g. small cell, non-small cell,squamous, adenocarcinoma), breast, thyroid, lymphoid, gastrointestinal,genito-urinary tract, kidney, bladder, liver (e.g. hepatocellularcancer), pancreas, ovary, cervix, endometrium, uterine, prostate, brain,as well as adenocarcinomas which include malignancies such as most coloncancers, colorectal cancer, renal-cell carcinoma, prostate cancer and/ortesticular tumors, non-small cell carcinoma of the lung, cancer of thesmall intestine and cancer of the esophagus.

The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. In some embodiments, thedisease is renal carcinoma or melanoma. Exemplary carcinomas includethose forming from tissue of the cervix, lung, prostate, breast, headand neck, colon and ovary. The term also includes carcinosarcomas, e.g.,which include malignant tumors composed of carcinomatous and sarcomatoustissues. An “adenocarcinoma” refers to a carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures.

The term “sarcoma” is art recognized and refers to malignant tumors ofmesenchymal derivation.

Additional examples of proliferative disorders include hematopoieticneoplastic disorders. As used herein, the term “hematopoietic neoplasticdisorders” includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias, e.g., erythroblasticleukemia and acute megakaryoblastic leukemia. Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. inOncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

In some embodiments, specific cancers that can be treated using themethods described herein are listed in Table 6 or 9, for example, andinclude, but are not limited to: breast, lung, prostate, CNS (e.g.,glioma), salivary gland, prostate, ovarian, and leukemias (e.g., ALL,CML, or AML). Associations of these genes with a particular cancer areknown in the art, e.g., as described in Futreal et al., Nat Rev Cancer.2004;4;177-83 (see, e.g., Table 1, incorporated by reference herein);and The COSMIC (Catalogue of Somatic Mutations in Cancer) database andwebsite, Bamford et al., Br J Cancer. 2004;91;355-8; see also Forbes etal., Curr Protoc Hum Genet. 2008;Chapter 10;Unit 10.11, and the COSMICdatabase, e.g., v.50 (Nov. 30, 2010). It is understood that reference toany particular type of cancer in, for example, Table 6 or 9 means thatpatients with other types of cancer, i.e., cancer in general, may betreated.

In addition, the methods described herein can be used for modulating(e.g., enhancing or decreasing) pluripotency of a stem cell and todirect stem cells down specific differentiation pathways to makeendoderm, mesoderm, ectoderm, and their developmental derivatives. Toincrease, maintain, or enhance pluripotency, the methods includeintroducing into the cell an inhibitory nucleic acid that specificallybinds to, or is complementary to, a long non-coding RNA as set forth inTable 3. To decrease pluripotency or enhance differentiation of a stemcell, the methods include introducing into the cell a long non-codingRNA as set forth in Table 3. Stem cells useful in the methods describedherein include adult stem cells (e.g., adult stem cells obtained fromthe inner ear, bone marrow, mesenchyme, skin, fat, liver, muscle, orblood of a subject, e.g., the subject to be treated); embryonic stemcells, or stem cells obtained from a placenta or umbilical cord;progenitor cells (e.g., progenitor cells derived from the inner ear,bone marrow, mesenchyme, skin, fat, liver, muscle, or blood); andinduced pluripotent stem cells (e.g., iPS cells).

In some embodiments, the methods described herein include administeringa composition, e.g., a sterile composition, comprising an inhibitorynucleic acid that is complementary to an lncRNA described herein, e.g.,as set forth in Table 1, 2, 3, 6, or 7, or Table 8. Inhibitory nucleicacids for use in practicing the methods described herein can be anantisense or small interfering RNA, including but not limited to anshRNA or siRNA. In some embodiments, the inhibitory nucleic acid is amodified nucleic acid polymer (e.g., a locked nucleic acid (LNA)molecule).

Inhibitory nucleic acids have been employed as therapeutic moieties inthe treatment of disease states in animals, including humans. Inhibitorynucleic acids can be useful therapeutic modalities that can beconfigured to be useful in treatment regimes for the treatment of cells,tissues and animals, especially humans.

For therapeutics, an animal, preferably a human, suspected of havingcancer is treated by administering an lncRNA or inhibitory nucleic acidin accordance with this invention. For example, in one non-limitingembodiment, the methods comprise the step of administering to the animalin need of treatment, a therapeutically effective amount of an lncRNA orinhibitory nucleic acid as described herein.

Inhibitory Nucleic Acids

Inhibitory nucleic acids useful in the present methods and compositionsinclude antisense oligonucleotides, ribozymes, external guide sequence(EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, moleculescomprising modified bases, locked nucleic acid molecules (LNAmolecules), antagomirs, peptide nucleic acid molecules (PNA molecules),and other oligomeric compounds or oligonucleotide mimetics whichhybridize to at least a portion of the target nucleic acid and modulateits function. In some embodiments, the inhibitory nucleic acids includeantisense RNA, antisense DNA, chimeric antisense oligonucleotides,antisense oligonucleotides comprising modified linkages, interferenceRNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA(miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA);small RNA-induced gene activation (RNAa); small activating RNAs(saRNAs), or combinations thereof. See, e.g., WO 2010040112.

In some embodiments, the inhibitory nucleic acids are 10 to 50, 13 to50, or 13 to 30 nucleotides in length. One having ordinary skill in theart will appreciate that this embodies oligonucleotides having antisense(complementary) portions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length,or any range therewithin. It is understood that non-complementary basesmay be included in such inhibitory nucleic acids; for example, aninhibitory nucleic acid 30 nucleotides in length may have a portion of15 bases that is complementary to the targeted RNA. In some embodiments,the oligonucleotides are 15 nucleotides in length. In some embodiments,the antisense or oligonucleotide compounds of the invention are 12 or 13to 30 nucleotides in length. One having ordinary skill in the art willappreciate that this embodies inhibitory nucleic acids having antisense(complementary) portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or any rangetherewithin.

Preferably the inhibitory nucleic acid comprises one or moremodifications comprising: a modified sugar moiety, and/or a modifiedinternucleoside linkage, and/or a modified nucleotide and/orcombinations thereof. It is not necessary for all positions in a givenoligonucleotide to be uniformly modified, and in fact more than one ofthe modifications described herein may be incorporated in a singleoligonucleotide or even at within a single nucleoside within anoligonucleotide.

In some embodiments, the inhibitory nucleic acids are chimericoligonucleotides that contain two or more chemically distinct regions,each made up of at least one nucleotide. These oligonucleotidestypically contain at least one region of modified nucleotides thatconfers one or more beneficial properties (such as, for example,increased nuclease resistance, increased uptake into cells, increasedbinding affinity for the target) and a region that is a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimericinhibitory nucleic acids of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures comprise, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference.

In some embodiments, the inhibitory nucleic acid comprises at least onenucleotide modified at the 2′ position of the sugar, most preferably a2′-O-alkyl, 2′-O-alkyl-O-alkyl or 2′-fluoro-modified nucleotide. Inother preferred embodiments, RNA modifications include 2′-fluoro,2′-amino and 2′ O-methyl modifications on the ribose of pyrimidines,abasic residues or an inverted base at the 3′ end of the RNA. Suchmodifications are routinely incorporated into oligonucleotides and theseoligonucleotides have been shown to have a higher Tm (i.e., highertarget binding affinity) than; 2′-deoxyoligonucleotides against a giventarget.

A number of nucleotide and nucleoside modifications have been shown tomake the oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligodeoxynucleotide; thesemodified oligos survive intact for a longer time than unmodifiedoligonucleotides. Specific examples of modified oligonucleotides includethose comprising modified backbones, for example, phosphorothioates,phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. Most preferred are oligonucleotides withphosphorothioate backbones and those with heteroatom backbones,particularly CH2-NH—O—CH2, CH, ˜N(CH3)˜O˜CH2 (known as amethylene(methylimino) or MMI backbone], CH2-O—N (CH3)-CH2, CH2-N(CH3)-N (CH3)-CH2 and O—N (CH3)-CH2 -CH2 backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH,); amide backbones(see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholinobackbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506);peptide nucleic acid (PNA) backbone (wherein the phosphodiester backboneof the oligonucleotide is replaced with a polyamide backbone, thenucleotides being bound directly or indirectly to the aza nitrogen atomsof the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497).Phosphorus-containing linkages include, but are not limited to,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates comprising 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates comprising 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2; see U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5, 177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Dwaine A. Braaschand David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis,volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214;Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc.Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506,issued Jul. 23, 1991. In some embodiments, the morpholino-basedoligomeric compound is a phosphorodiamidate morpholino oligomer (PMO)(e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001;and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures ofwhich are incorporated herein by reference in their entireties).

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., J. Am. Chem. Soc., 2000, 122, 8595-8602.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, each of which is herein incorporated by reference.

Modified oligonucleotides are also known that include oligonucleotidesthat are based on or constructed from arabinonucleotide or modifiedarabinonucleotide residues. Arabinonucleosides are stereoisomers ofribonucleosides, differing only in the configuration at the 2′-positionof the sugar ring. In some embodiments, a 2′-arabino modification is2′-F arabino. In some embodiments, the modified oligonucleotide is2′-fluoro-D-arabinonucleic acid (FANA) (as described in, for example,Lon et al., Biochem., 41:3457-3467, 2002 and Min et al., Bioorg. Med.Chem. Lett., 12:2651-2654, 2002; the disclosures of which areincorporated herein by reference in their entireties). Similarmodifications can also be made at other positions on the sugar,particularly the 3′ position of the sugar on a 3′ terminal nucleoside orin 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminalnucleotide.

PCT Publication No. WO 99/67378 discloses arabinonucleic acids (ANA)oligomers and their analogues for improved sequence specific inhibitionof gene expression via association to complementary messenger RNA.

Other preferred modifications include ethylene-bridged nucleic acids(ENAs) (e.g., International Patent Publication No. WO 2005/042777,Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al.,Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther.,8:144-149, 2006 and Hone et al., Nucleic Acids Symp. Ser (Oxf),49:171-172, 2005; the disclosures of which are incorporated herein byreference in their entireties). Preferred ENAs include, but are notlimited to, 2′-O,4′-C-ethylene-bridged nucleic acids.

Examples of LNAs are described in WO/2008/043753 and include compoundsof the following formula.

where X and Y are independently selected among the groups —O—,

—S—, —N(H)—, N(R)—, —CH2- or —CH— (if part of a double bond),

—CH₂—O—, —CH₂—S—, —CH₂—N(H)—, —CH₂—N(R)—, —CH₂—CH₂— or —CH₂—CH— (if partof a double bond),

—CH═CH-, where R is selected from hydrogen and C₁₋₄-alkyl; Z and Z* areindependently selected among an internucleoside linkage, a terminalgroup or a protecting group; B constitutes a natural or non-naturalnucleotide base moiety; and the asymmetric groups may be found in eitherorientation.

Preferably, the LNA used in the oligomer of the invention comprises atleast one LNA unit according any of the formulas

wherein Y is —O—, —S—, —NH—, or N(R^(H)); Z and Z* are independentlyselected among an internucleoside linkage, a terminal group or aprotecting group; B constitutes a natural or non-natural nucleotide basemoiety, and RH is selected from hydrogen and C₁₋₄-alkyl.

Preferably, the Locked Nucleic Acid (LNA) used in the oligomericcompound, such as an antisense oligonucleotide, of the inventioncomprises at least one nucleotide comprises a Locked Nucleic Acid (LNA)unit according any of the formulas shown in Scheme 2 ofPCT/DK2006/000512.

Preferably, the LNA used in the oligomer of the invention comprisesinternucleoside linkages selected from -0-P(O)₂—O—, —O—P(O,S)—O—,-0-P(S)₂—O—, —S—P(0)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—, -0-P(O)₂—S—,—O—P(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—, O—PO(OCH₃)—O—,—O—PO(NR^(H))—O—, -0-PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—,where R^(H) is selected from hydrogen and C₁₋₄-alkyl.

Specifically preferred LNA units are shown in scheme 2:

The term “thio-LNA” comprises a locked nucleotide in which at least oneof X or Y in the general formula above is selected from S or —CH2-S—.Thio-LNA can be in both beta-D and alpha-L-configuration.

The term “amino-LNA” comprises a locked nucleotide in which at least oneof X or Y in the general formula above is selected from —N(H)—, N(R)—,CH₂—N(H)—, and —CH₂—N(R)— where R is selected from hydrogen andC₁₋₄-alkyl. Amino-LNA can be in both beta-D and alpha-L-configuration.

The term “oxy-LNA” comprises a locked nucleotide in which at least oneof X or Y in the general formula above represents —O— or —CH₂—O—.Oxy-LNA can be in both beta-D and alpha-L-configuration.

The term “ena-LNA” comprises a locked nucleotide in which Y in thegeneral formula above is —CH₂—O— (where the oxygen atom of —CH₂—O— isattached to the 2′-position relative to the base B).

LNAs are described in additional detail below.

One or more substituted sugar moieties can also be included, e.g., oneof the following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃ OCH₃,OCH₃ O(CH₂)n CH₃, O(CH₂)n NH₂ or O(CH₂)n CH₃ where n is from 1 to about10; Ci to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl,alkaryl or aralkyl; Cl; Br; CN; CF3 ; OCF3; O—, S—, or N-alkyl; O—, S—,or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. A preferredmodification includes 2′-methoxyethoxy [2′-0-CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl)] (Martin et al, HeIv. Chim. Acta, 1995, 78, 486).Other preferred modifications include 2′-methoxy (2′-0-CH₃), 2′-propoxy(2′-OCH₂ CH₂CH₃) and 2′-fluoro (2′-F). Similar modifications may also bemade at other positions on the oligonucleotide, particularly the 3′position of the sugar on the 3′ terminal nucleotide and the 5′ positionof 5′ terminal nucleotide. Oligonucleotides may also have sugar mimeticssuch as cyclobutyls in place of the pentofuranosyl group.

Inhibitory nucleic acids can also include, additionally oralternatively, nucleobase (often referred to in the art simply as“base”) modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include adenine (A), guanine (G), thymine (T),cytosine (C) and uracil (U). Modified nucleobases include nucleobasesfound only infrequently or transiently in natural nucleic acids, e.g.,hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly5-methylcytosine (also referred to as 5-methyl-2′ deoxycytosine andoften referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC),glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, aswell as synthetic nucleobases, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,5-propynyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, 6-aminopurine, 2-aminopurine,2-chloro-6-aminopurine and 2,6-diaminopurine or other diaminopurines.See, e.g., Kornberg, “DNA Replication,” W. H. Freeman & Co., SanFrancisco, 1980, pp75-'7′7; and Gebeyehu, G., et al. Nucl. Acids Res.,15:4513 (1987)). A “universal” base known in the art, e.g., inosine, canalso be included. 5-Me-C substitutions have been shown to increasenucleic acid duplex stability by 0.6-1.2<0>C. (Sanghvi, in Crooke, andLebleu, eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the modificationsdescribed herein may be incorporated in a single oligonucleotide or evenat within a single nucleoside within an oligonucleotide.

In some embodiments, both a sugar and an internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, anoligonucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide isreplaced with an amide containing backbone, for example, anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al, Science, 1991, 254, 1497-1500.

Inhibitory nucleic acids can also include one or more nucleobase (oftenreferred to in the art simply as “base”) modifications or substitutions.As used herein, “unmodified” or “natural” nucleobases comprise thepurine bases adenine (A) and guanine (G), and the pyrimidine basesthymine (T), cytosine (C) and uracil (U). Modified nucleobases compriseother synthetic and natural nucleobases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylquanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further, nucleobases comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in “The Concise Encyclopedia of PolymerScience And Engineering”, pages 858-859, Kroschwitz, ed. John Wiley &Sons, 1990;, those disclosed by Englisch et al., Angewandle Chemie,International Edition, 1991, 30, page 613, and those disclosed bySanghvi, Chapter 15, Antisense Research and Applications,” pages289-302, Crooke, and Lebleu, eds., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, comprising 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2<0>C (Sanghvi, etal., eds, “Antisense Research and Applications,” CRC Press, Boca Raton,1993, pp. 276-278) and are presently preferred base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications. Modified nucleobases are described in U.S. Pat. Nos.3,687,808, as well as 4,845,205; 5,130,302; 5,134,066; 5,175, 273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177;5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and5,681,941, each of which is herein incorporated by reference.

In some embodiments, the inhibitory nucleic acids are chemically linkedto one or more moieties or conjugates that enhance the activity,cellular distribution, or cellular uptake of the oligonucleotide. Forexample, one or more inhibitory nucleic acids, of the same or differenttypes, can be conjugated to each other; or inhibitory nucleic acids canbe conjugated to targeting moieties with enhanced specificity for a celltype or tissue type. Such moieties include, but are not limited to,lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al, Ann. N. Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al.,Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-toxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996,277, 923-937). See also U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082, 830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5, 245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391, 723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941, each of which is herein incorporated byreference.

These moieties or conjugates can include conjugate groups covalentlybound to functional groups such as primary or secondary hydroxyl groups.Conjugate groups of the invention include intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, polyethers,groups that enhance the pharmacodynamic properties of oligomers, andgroups that enhance the pharmacokinetic properties of oligomers. Typicalconjugate groups include cholesterols, lipids, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes.

Groups that enhance the pharmacodynamic properties, in the context ofthis invention, include groups that improve uptake, enhance resistanceto degradation, and/or strengthen sequence-specific hybridization withthe target nucleic acid. Groups that enhance the pharmacokineticproperties, in the context of this invention, include groups thatimprove uptake, distribution, metabolism or excretion of the compoundsof the present invention. Representative conjugate groups are disclosedin International Patent Application No. PCT/US92/09196, filed Oct. 23,1992, and U.S. Pat. No. 6,287,860, which are incorporated herein byreference. Conjugate moieties include, but are not limited to, lipidmoieties such as a cholesterol moiety, cholic acid, a thioether, e.g.,hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol ortriethylammoniuml,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, apolyamine or a polyethylene glycol chain, or adamantane acetic acid, apalmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941.

The inhibitory nucleic acids useful in the present methods aresufficiently complementary to the target lncRNA, e.g., hybridizesufficiently well and with sufficient biological functional specificity,to give the desired effect. “Complementary” refers to the capacity forpairing, through base stacking and specific hydrogen bonding, betweentwo sequences comprising naturally or non-naturally occurring (e.g.,modified as described above) bases (nucleosides) or analogs thereof. Forexample, if a base at one position of an inhibitory nucleic acid iscapable of hydrogen bonding with a base at the corresponding position ofa lncRNA, then the bases are considered to be complementary to eachother at that position. 100% complementarity is not required. As notedabove, inhibitory nucleic acids can comprise universal bases, or inertabasic spacers that provide no positive or negative contribution tohydrogen bonding. Base pairings may include both canonical Watson-Crickbase pairing and non-Watson-Crick base pairing (e.g., Wobble basepairing and Hoogsteen base pairing). It is understood that forcomplementary base pairings, adenosine-type bases (A) are complementaryto thymidine-type bases (T) or uracil-type bases (U), that cytosine-typebases (C) are complementary to guanosine-type bases (G), and thatuniversal bases such as 3-nitropyrrole or 5-nitroindole can hybridize toand are considered complementary to any A, C, U, or T. Nichols et al.,Nature, 1994;369:492-493 and Loakes et al., Nucleic Acids Res.,1994;22:4039-4043.

Inosine (I) has also been considered in the art to be a universal baseand is considered complementary to any A, C, U or T. See Watkins andSantaLucia, Nucl. Acids Research, 2005; 33 (19): 6258-6267.

In some embodiments, the location on a target lncRNA to which aninhibitory nucleic acids hybridizes is defined as a target region towhich a protein binding partner binds. These regions can be identifiedby reviewing the data submitted herewith in Appendix I and identifyingregions that are enriched in the dataset; these regions are likely toinclude the protein binding sequences. The identification of suchregions, termed Peaks, is described in Example 8 below. Routine methodscan be used to design an inhibitory nucleic acid that binds to thissequence with sufficient specificity. In some embodiments, the methodsinclude using bioinformatics methods known in the art to identifyregions of secondary structure, e.g., one, two, or more stem-loopstructures, or pseudoknots, and selecting those regions to target withan inhibitory nucleic acid.

While the specific sequences of certain exemplary target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional target segments are readilyidentifiable by one having ordinary skill in the art in view of thisdisclosure. Target segments 5-500 nucleotides in length comprising astretch of at least five (5) consecutive nucleotides within the proteinbinding region, or immediately adjacent thereto, are considered to besuitable for targeting as well. Target segments can include sequencesthat comprise at least the 5 consecutive nucleotides from the5′-terminus of one of the protein binding regions (the remainingnucleotides being a consecutive stretch of the same RNA beginningimmediately upstream of the 5′-terminus of the binding segment andcontinuing until the inhibitory nucleic acid contains about 5 to about100 nucleotides). Similarly preferred target segments are represented byRNA sequences that comprise at least the 5 consecutive nucleotides fromthe 3′-terminus of one of the illustrative preferred target segments(the remaining nucleotides being a consecutive stretch of the samelncRNA beginning immediately downstream of the 3′-terminus of the targetsegment and continuing until the inhibitory nucleic acid contains about5 to about 100 nucleotides). One having skill in the art armed with thesequences provided herein will be able, without undue experimentation,to identify further preferred protein binding regions to target withcomplementary inhibitory nucleic acids.

In the context of the present disclosure, hybridization means basestacking and hydrogen bonding, which may be Watson-Crick, Hoogsteen orreversed Hoogsteen hydrogen bonding, between complementary nucleoside ornucleotide bases. For example, adenine and thymine are complementarynucleobases which pair through the formation of hydrogen bonds.Complementary, as the term is used in the art, refers to the capacityfor precise pairing between two nucleotides. For example, if anucleotide at a certain position of an oligonucleotide is capable ofhydrogen bonding with a nucleotide at the same position of a lncRNAmolecule, then the inhibitory nucleic acid and the lncRNA are consideredto be complementary to each other at that position. The inhibitorynucleic acids and the lncRNA are complementary to each other when asufficient number of corresponding positions in each molecule areoccupied by nucleotides that can hydrogen bond with each other throughtheir bases. Thus, “specifically hybridizable” and “complementary” areterms which are used to indicate a sufficient degree of complementarityor precise pairing such that stable and specific binding occurs betweenthe inhibitory nucleic acid and the lncRNA target. For example, if abase at one position of an inhibitory nucleic acid is capable ofhydrogen bonding with a base at the corresponding position of a lncRNA,then the bases are considered to be complementary to each other at thatposition. 100% complementarity is not required.

It is understood in the art that a complementary nucleic acid sequenceneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. A complementary nucleic acid sequence forpurposes of the present methods is specifically hybridizable whenbinding of the sequence to the target lncRNA molecule interferes withthe normal function of the target lncRNA to cause a loss of activity(e.g., inhibiting PRC2-associated repression with consequentup-regulation of gene expression) and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the sequence tonon-target lncRNA sequences under conditions in which avoidance ofnon-specific binding is desired, e.g., under physiological conditions inthe case of in vivo assays or therapeutic treatment, and in the case ofin vitro assays, under conditions in which the assays are performedunder suitable conditions of stringency. For example, stringent saltconcentration will ordinarily be less than about 750 mM NaCl and 75 mMtrisodium citrate, preferably less than about 500 mM NaCl and 50 mMtrisodium citrate, and more preferably less than about 250 mM NaCl and25 mM trisodium citrate. Low stringency hybridization can be obtained inthe absence of organic solvent, e.g., formamide, while high stringencyhybridization can be obtained in the presence of at least about 35%formamide, and more preferably at least about 50% formamide. Stringenttemperature conditions will ordinarily include temperatures of at leastabout 30° C., more preferably of at least about 37° C., and mostpreferably of at least about 42° C. Varying additional parameters, suchas hybridization time, the concentration of detergent, e.g., sodiumdodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA,are well known to those skilled in the art. Various levels of stringencyare accomplished by combining these various conditions as needed. In apreferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl,75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment,hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

In general, the inhibitory nucleic acids useful in the methods describedherein have at least 80% sequence complementarity to a target regionwithin the target nucleic acid, e.g., 90%, 95%, or 100% sequencecomplementarity to the target region within an lncRNA. For example, anantisense compound in which 18 of 20 nucleobases of the antisenseoligonucleotide are complementary, and would therefore specificallyhybridize, to a target region would represent 90 percentcomplementarity. Percent complementarity of an inhibitory nucleic acidwith a region of a target nucleic acid can be determined routinely usingbasic local alignment search tools (BLAST programs) (Altschul et al., J.Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7,649-656). Antisense and other compounds of the invention that hybridizeto an lncRNA are identified through routine experimentation. In generalthe inhibitory nucleic acids must retain specificity for their target,i.e., either do not directly bind to, or do not directly significantlyaffect expression levels of, transcripts other than the intended target.

Target-specific effects, with corresponding target-specific functionalbiological effects, are possible even when the inhibitory nucleic acidexhibits non-specific binding to a large number of non-target RNAs. Forexample, short 8 base long inhibitory nucleic acids that are fullycomplementary to a lncRNA may have multiple 100% matches to hundreds ofsequences in the genome, yet may produce target-specific effects, e.g.upregulation of a specific target gene through inhibition of PRC2activity. 8-base inhibitory nucleic acids have been reported to preventexon skipping with with a high degree of specificity and reducedoff-target effect. See Singh et al., RNA Biol., 2009; 6(3): 341-350.8-base inhibitory nucleic acids have been reported to interfere withmiRNA activity without significant off-target effects. See Obad et al.,Nature Genetics, 2011; 43: 371-378.

For further disclosure regarding inhibitory nucleic acids, please seeUS2010/0317718 (antisense oligos); US2010/0249052 (double-strandedribonucleic acid (dsRNA)); US2009/0181914 and US2010/0234451 (LNAmolecules); US2007/0191294 (siRNA analogues); US2008/0249039 (modifiedsiRNA); and WO010/129746 and WO2010/040112 (inhibitory nucleic acids).

Antisense

In some embodiments, the inhibitory nucleic acids are antisenseoligonucleotides.

Antisense oligonucleotides are typically designed to block expression ofa DNA or RNA target by binding to the target and halting expression atthe level of transcription, translation, or splicing. Antisenseoligonucleotides of the present invention are complementary nucleic acidsequences designed to hybridize under stringent conditions to an lncRNAin vitro, and are expected to inhibit the activity of PRC2 in vivo.Thus, oligonucleotides are chosen that are sufficiently complementary tothe target, i.e., that hybridize sufficiently well and with sufficientbiological functional specificity, to give the desired effect.

Modified Base, including Locked Nucleic Acids (LNAs)

In some embodiments, the inhibitory nucleic acids used in the methodsdescribed herein comprise one or more modified bonds or bases. Modifiedbases include phosphorothioate, methylphosphonate, peptide nucleicacids, or locked nucleic acids (LNAs). Preferably, the modifiednucleotides are part of locked nucleic acid molecules, including[alpha]-L-LNAs. LNAs include ribonucleic acid analogues wherein theribose ring is “locked” by a methylene bridge between the 2′-oxgygen andthe 4′-carbon—i.e., oligonucleotides containing at least one LNAmonomer, that is, one 2′-O,4′-C-methylene-β-D-ribofuranosyl nucleotide.LNA bases form standard Watson-Crick base pairs but the lockedconfiguration increases the rate and stability of the basepairingreaction (Jepsen et al., Oligonucleotides, 14, 130-146 (2004)). LNAsalso have increased affinity to base pair with RNA as compared to DNA.These properties render LNAs especially useful as probes forfluorescence in situ hybridization (FISH) and comparative genomichybridization, as knockdown tools for miRNAs, and as antisenseoligonucleotides to target mRNAs or other RNAs, e.g., lncRNAs asdescribed herien.

The modified base/LNA molecules can include molecules comprising 10-30,e.g., 12-24, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of thestrands is substantially identical, e.g., at least 80% (or more, e.g.,85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatchednucleotide(s), to a target region in the lncRNA. The modified base/LNAmolecules can be chemically synthesized using methods known in the art.

The modified base/LNA molecules can be designed using any method knownin the art; a number of algorithms are known, and are commerciallyavailable (e.g., on the internet, for example at exiqon.com). See, e.g.,You et al., Nuc. Acids. Res. 34:e60 (2006); McTigue et al., Biochemistry43:5388-405 (2004); and Levin et al., Nuc. Acids.

Res. 34:e142 (2006). For example, “gene walk” methods, similar to thoseused to design antisense oligos, can be used to optimize the inhibitoryactivity of a modified base/LNA molecule; for example, a series ofoligonucleotides of 10-30 nucleotides spanning the length of a targetlncRNA can be prepared, followed by testing for activity. Optionally,gaps, e.g., of 5-10 nucleotides or more, can be left between the LNAs toreduce the number of oligonucleotides synthesized and tested. GC contentis preferably between about 30-40%. General guidelines for designingmodified base/LNA molecules are known in the art; for example, LNAsequences will bind very tightly to other LNA sequences, so it ispreferable to avoid significant complementarity within an LNA molecule.Contiguous runs of three or more Gs or Cs, or more than four LNAresidues, should be avoided where possible (for example, it may not bepossible with very short (e.g., about 9-10 nt) oligonucleotides). Insome embodiments, the LNAs are xylo-LNAs.

In some embodiments, the modified base/LNA molecules can be designed totarget a specific region of the lncRNA. For example, a specificfunctional region can be targeted, e.g., a region comprising a known RNAlocalization motif (i.e., a region complementary to the target nucleicacid on which the lncRNA acts), or a region comprising a known proteinbinding region, e.g., a Polycomb (e.g., Polycomb Repressive Complex 2(PRC2), comprised of H3K27 methylase EZH2, SUZ12, and EED)) orLSD1/CoREST/REST complex binding region (see, e.g., Tsai et al.,Science. 2010 Aug. 6; 329(5992):689-93. Epub 2010 Jul. 8; and Zhao etal., Science. 2008 Oct. 31; 322(5902):750-6). Sarma et al., “Lockednucleic acids (LNAs) reveal sequence requirements and kinetics of XistRNA localization to the X chromosome.” PNAS published ahead of printDec. 6, 2010, doi:10.1073/pnas.1009785107. Alternatively or in addition,highly conserved regions can be targeted, e.g., regions identified byaligning sequences from disparate species such as primate (e.g., human)and rodent (e.g., mouse) and looking for regions with high degrees ofidentity. Percent identity can be determined routinely using basic localalignment search tools (BLAST programs) (Altschul et al., J. Mol. Biol.,1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656),e.g., using the default parameters.

For additional information regarding LNA molecules see U.S. Pat. Nos.6,268,490; 6,734,291; 6,770,748; 6,794,499; 7,034,133; 7,053,207;7,060,809; 7,084,125; and 7,572,582; and U.S. Pre-Grant Pub. Nos.20100267018; 20100261175; and 20100035968; Koshkin et al. Tetrahedron54, 3607-3630 (1998); Obika et al. Tetrahedron Lett. 39, 5401-5404(1998); Jepsen et al., Oligonucleotides 14:130-146 (2004); Kauppinen etal., Drug Disc. Today 2(3):287-290 (2005); and Ponting et al., Cell136(4):629-641 (2009), and references cited therein.

In a related aspect, the present disclosure demonstrates the ability ofLNA molecules to displace a cis-acting nuclear long ncRNA with fastkinetics (e.g., RNA/PRC2 disassociation from the chromosome after 2, 5,10 seconds up to 60 minutes as described herein)—a property that enablesthe modification and study of the function of long ncRNAs in ways notpreviously possible. Using 17 kb Xist RNA as a model, the presentinventors showed that LNA molecules designed to specifically target thetranscript leads to extremely rapid displacement of the RNA from theinactive X-chromosome. Interestingly, while the RNA is displaced,transcript stability is not affected. Targeting different Xist regionshas allowed the identification of a localization domain and show thatPolycomb repressive complex 2 (PRC2) is displaced together with Xist.Thus, PRC2 depends on RNA for both initial targeting to and stableassociation with chromatin. Time-course analysis of RNA relocalizationsuggests that Xist and PRC2 spread along X at the same time but does notreach saturating levels for 24 hours, providing a window of opportunityto reprogram the chromatin, if necessary.

It is remarkable that targeting a small region within a 17-kb RNA couldproduce such dramatic effects. The rapid effects suggest that the XistRNA-protein complex may be anchored to the inactive X chromosome (Xi)chromatin via Repeat C. Alternatively, the LNA molecule's binding toRepeat C could change RNA conformation and interfere with a remoteanchoring domain. While RNA displacement occurs with rapid kinetics, therecovery period is prolonged. Although full Xist clouds are restoredwithin 8 hours, the full complement of PRC2 is not recovered for up to24 hours. This implies that, during the spread of X-chromosomeinactivation (XCI), synthesis of the RNA is not the rate-limiting step;rather, it is the recruitment of associated silencing proteins such asPRC2. The rapid displacement of Xist and the slow kinetics of recoveryprovided a large window of opportunity to investigate Xist's spreadingpattern relative to that of PRC2. Time-course analysis during therecovery phase indicates that Xist RNA binds most strongly near the Xistlocus at first but spreads to the rest of Xi at the same time.Similarly, PRC2 is recruited synchronously throughout the X.Interestingly, neither Xist nor PRC2 levels reach saturationimmediately, as the coating of Xist is not complete until t=8 hr andbinding of PRC2 does not peak until t=24 hr. Combined, this analysisimplies that establishment of chromosome-wide silencing may berelatively slow.

As demonstrated herein, LNA molecules can be used as a valuable tool tomanipulate and aid analysis of long nuclear ncRNAs. Advantages offeredby an LNA molecule-based system are the relatively low costs, easydelivery, and rapid action. While other inhibitory nucleic acids mayexhibit effects after longer periods of time, LNA molecules exhibiteffects that are more rapid, e.g., a comparatively early onset ofactivity, are fully reversible after a recovery period following thesynthesis of new lncRNA, and occur without causing substantial orsubstantially complete RNA cleavage or degradation. One or more of thesedesign properties may be desired properties of the inhibitory nucleicacids of the invention. Additionally, LNA molecules make possible thesystematic targeting of domains within much longer nuclear transcripts.Although a PNA-based system has been described earlier, the effects onXi were apparent only after 24 hours (13). The LNA technology enableshigh-throughput screens for functional analysis of long non-coding RNAsand also provides a novel tool to manipulate chromatin states in vivofor therapeutic applications.

In various related aspects, the methods described herein include usingLNA molecules to target lncRNAs for a number of uses, including as aresearch tool to probe the function of a specific lncRNA, e.g., in vitroor in vivo. The methods include selecting one or more desired lncRNAs,designing one or more LNA molecules that target the lncRNA, providingthe designed LNA molecule, and administering the LNA molecule to a cellor animal. The methods can optionally include selecting a region of thelncRNA and designing one or more LNA molecules that target that regionof the lncRNA.

Aberrant imprinted gene expression is implicated in several diseasesincluding Long QT syndrome, Beckwith-Wiedemann, Prader-Willi, andAngelman syndromes, as well as behavioral disorders and carcinogenesis(see, e.g., Falls et al., Am. J. Pathol. 154:635-647 (1999); Lalande,Annu Rev Genet 30:173-195 (1996); Hall Annu Rev Med. 48:35-44 (1997)).LNA molecules can be created to treat such imprinted diseases. As oneexample, the long QT Syndrome can be caused by a K+gated Calcium-channelencoded by Kcnql. This gene is regulated by its antisense counterpart,the long noncoding RNA, Kcnqlotl (Pandey et al., Mol Cell. 2008 Oct. 24;32(2):232-46). Disease arises when Kcnqlotl is aberrantly expressed. LNAmolecules can be created to downregulate Kcnqlotl, thereby restoringexpression of Kcnql. As another example, LNA molecules could inhibitLncRNA cofactors for polycomb complex chromatin modifiers to reverse theimprinted defect.

From a commercial and clinical perspective, the timepoints between about1 to 24 hours potentially define a window for epigenetic reprogramming.The advantage of the LNA system is that it works quickly, with a definedhalf-life, and is therefore reversible upon degradation of LNAs, at thesame time that it provides a discrete timeframe during which epigeneticmanipulations can be made. By targeting nuclear long ncRNAs, LNAmolecules or similar polymers, e.g., xylo-LNAs, might be utilized tomanipulate the chromatin state of cells in culture or in vivo, bytransiently eliminating the regulatory RNA and associated proteins longenough to alter the underlying locus for therapeutic purposes. Inparticular, LNA molecules or similar polymers that specifically bind to,or are complementary to, PRC2-binding lncRNA can prevent recruitment ofPRC2 to a specific chromosomal locus, in a gene-specific fashion.

LNA molecules might also be administered in vivo to treat other humandiseases, such as but not limited to cancer, neurological disorders,infections, inflammation, and myotonic dystrophy. For example, LNAmolecules might be delivered to tumor cells to downregulate the biologicactivity of a growth-promoting or oncogenic long nuclear ncRNA (e.g.,Gtl2 or MALAT1 (Luo et al., Hepatology. 44(4):1012-24 (2006)), a lncRNAassociated with metastasis and is frequently upregulated in cancers).Repressive lncRNAs downregulating tumor suppressors can also be targetedby LNA molecules to promote reexpression. For example, expression of theINK4b/ARF/INK4a tumor suppressor locus is controlled by Polycomb groupproteins including PRC1 and PRC2 and repressed by the antisensenoncoding RNA ANRIL (Yap et al., Mol Cell. 2010 Jun. 11; 38(5):662-74).ANRIL can be targeted by LNA molecules to promote reexpression of theINK4b/ARF/INK4a tumor suppressor. Some lncRNA may be positive regulatorsof oncogenes. Such “activating lncRNAs” have been described recently(e.g., Jpx (Tian et al., Cell. 143(3):390-403 (2010) and others (Ørom etal., Cell. 143(1):46-58 (2010)). Therefore, LNA molecules could bedirected at these activating lncRNAs to downregulate oncogenes. LNAmolecules could also be delivered to inflammatory cells to downregulateregulatory lncRNA that modulate the inflammatory or immune response.(e.g., LincRNA-Cox2, see Guttman et al., Nature. 458(7235):223-7. Epub2009 Feb. 1 (2009)).

In still other related aspects, the LNA molecules targeting lncRNAsdescribed herein can be used to create animal or cell models ofconditions associated with altered gene expression (e.g., as a result ofaltered epigenetics).

For example, it was first noticed about half a century ago thatX-chromosome changes are often seen in female reproductive cancers. Some70% of breast carcinomas lack a ‘Barr body’, the cytologic hallmark ofthe inactive X chromosome (Xi), and instead harbor two or more active Xs(Xa). Additional X's are also a risk factor for men, as XXY men(Klinefelter Syndrome) have a 20- to 50-fold increased risk of breastcancer in a BRCA1 background. The X is also known to harbor a number ofoncogenes. Supernumerary Xa's correlate with a poor prognosis and standas one of the most common cytogenetic abnormalities not only inreproductive cancers but also in leukemias, lymphomas, and germ celltumors of both sexes. See, e.g., Liao et al., Cancer Invest 21, 641-58(2003); Spatz et al., Nat Rev Cancer 4, 617-29 (2004); Barr et al., ProcCan Cancer Conf 2, 3-16 (1957); Borah et al., J Surg Oncol 13, 1-7(1980); Camargo and Wang, Hum Genet 55, 81-5 (1980); Dutrillaux et al.,Int J Cancer 38, 475-9 (1986); Ghosh and ShahCancer Genet Cytogenet 4,269-74 (1981); Ghosh and Shah, Med Hypotheses 7, 1099-104 (1981); Ghoshet al., Acta Cytol 27, 202-3 (1983); Huang et al., Mol Cancer Ther 1,769-76 (2002); Kawakami et al., Lancet 363, 40-2 (2004); Kawakami etal., J Urol 169, 1546-52 (2003); Kawakami et al., Oncogene 23, 6163-9(2004); Moore and Barr, Br J Cancer 9, 246-52 (1955); Moore and Barr, BrJ Cancer 11, 384-90 (1957); Moore et al., J Exp Zool 135, 101-25 (1957);Rosen et al., Ann Clin Lab Sci 7, 491-9 (1977); Sirchia et al., CancerRes 65, 2139-46 (2005); Tavares, Lancet 268, 948-9 (1955); Tavares,Medico (Porto) 12, 97-100 (1961); Tavares, Acta Cytol 6, 90-4 (1962);Wang et al., Cancer Genet Cytogenet 46, 271-80 (1990); and Ganesan etal., Cold Spring Harb Symp Quant Biol 70, 93-7 (2005).

Some 60% of childhood acute lymphoblastic leukemias (ALL) also displayextra X's; in chronic neutrophilic leukemia, the gain of X is sometimesthe only obvious abnormality and is associated with progression to blastcrisis (see, e.g., Heinonen and Mahlamaki, Cancer Genet Cytogenet 87,123-6 (1996); Heinonen et al., Med Pediatr Oncol 32, 360-5 (1999); andYamamoto et al., Cancer Genet Cytogenet 134, 84-7 (2002)). Theseobservations are so far only correlative but together hint that the Xmay be an accomplice in carcinogenesis. Xist, therefore may be a tumorsuppressor. Preliminary data obtained after deleting Xist in specificlineages in male and female mice shows that increased B cellproliferation occurs in a subset of mice (but not in controls; n=9).Without wishing to be bound by theory, one potential mechanism is thatloss of Xist in cells leads X-reactivation or Xa duplication, resultingin an XaXa state in the cell. The consequent increased expression ofX-oncogenes induces a pre-cancerous state, and an accumulation ofadditional epigenetic/genetic changes (e.g., genome-wide changes thenresults in cancer. Thus, Xist may be a tumor suppressor.

An animal model of specific cancers (e.g., those cancers known in theart and described above that are associated with X-chromosome changes)could be created by using an XIST-LNA, e.g., the XIST LNAs describedherein, to remove XIST in a cell or tissue and developmentally specificway.

The methods described herein may also be useful for creating animal orcell models of other conditions associated with aberrant imprinted geneexpression, e.g., as noted above.

In various related aspects, the results described herein demonstrate theutility of LNA molecules for targeting long ncRNA, for example, totransiently disrupt chromatin for purposes of reprogramming chromatinstates ex vivo. Because LNA molecules stably displace RNA for hours andchromatin does not rebuild for hours thereafter, LNA molecules create awindow of opportunity to manipulate the epigenetic state of specificloci ex vivo, e.g., for reprogramming of hiPS and hESC prior to stemcell therapy. For example, Gtl2 controls expression of DLK1, whichmodulates the pluripotency of iPS cells. Low Gtl2 and high DLK1 iscorrelated with increased pluripotency and stability in human iPS cells.Thus, LNA molecules targeting Gtl2 can be used to inhibitdifferentiation and increase pluripotency and stability of iPS cells.

See also U.S. Ser. No. 61/412,862, which is incorporated by referenceherein in its entirety.

Antagomirs

In some embodiments, the inhibitory nucleic acid is an antagomir.Antagomirs are chemically modified antisense oligonucleotides that cantarget an lncRNA. For example, an antagomir for use in the methodsdescribed herein can include a nucleotide sequence sufficientlycomplementary to hybridize to an lncRNA target sequence of about 12 to25 nucleotides, preferably about 15 to 23 nucleotides.

In some embodiments, antagomirs include a cholesterol moiety, e.g., atthe 3′-end. In some embodiments, antagomirs have various modificationsfor RNase protection and pharmacologic properties such as enhancedtissue and cellular uptake. For example, in addition to themodifications discussed above for antisense oligos, an antagomir canhave one or more of complete or partial 2′-O-methylation of sugar and/ora phosphorothioate backbone. Phosphorothioate modifications provideprotection against RNase or other nuclease activity and theirlipophilicity contributes to enhanced tissue uptake. In someembodiments, the antagomir cam include six phosphorothioate backbonemodifications;

two phosphorothioates are located at the 5′-end and four at the 3′-end,but other patterns of phosphorothioate modification are also commonlyemployed and effective. See, e.g., Krutzfeldt et al., Nature 438,685-689(2005); Czech, N Engl J Med 2006; 354:1194-1195 (2006); Robertson etal., Silence. 1:10 (2010); Marquez and McCaffrey, Hum Gene Ther.19(1):27-38 (2008); van Rooij et al., Circ Res. 103(9):919-928 (2008);and Liu et al., Int. J. Mol. Sci. 9:978-999 (2008). Krutzfeld et al.(2005) describe chemically engineered oligonucleotides, termed‘antagomirs’, that are reported to be are efficient and specificsilencers of endogenous miRNAs in mice.

In general, the design of an antagomir avoids target RNA degradation dueto the modified sugars present in the molecule. The presence of anunbroken string of unmodified sugars supports RNAseH recruitment andenzymatic activity. Thus, typically the design of an antagomir willinclude bases that contain modified sugar (e.g., LNA), at the ends orinterspersed with natural ribose or deoxyribose nucleobases.

Antagomirs useful in the present methods can also be modified withrespect to their length or otherwise the number of nucleotides making upthe antagomir. In some embodiments, the antagomirs must retainspecificity for their target, i.e., must not directly bind to, ordirectly significantly affect expression levels of, transcripts otherthan the intended target. In some embodiments antagomirs may exhibitnonspecific binding that does not produce significant undesired biologiceffect, e.g. the antagomirs do not affect expression levels ofnon-target transcripts or their association with regulatory proteins orregulatory RNAs.

Interfering RNA, including siRNA/shRNA

In some embodiments, the inhibitory nucleic acid sequence that iscomplementary to an lncRNA can be an interfering RNA, including but notlimited to a small interfering RNA (“siRNA”) or a small hairpin RNA(“shRNA”). Methods for constructing interfering RNAs are well known inthe art. For example, the interfering RNA can be assembled from twoseparate oligonucleotides, where one strand is the sense strand and theother is the antisense strand, wherein the antisense and sense strandsare self-complementary (i.e., each strand comprises nucleotide sequencethat is complementary to nucleotide sequence in the other strand; suchas where the antisense strand and sense strand form a duplex or doublestranded structure); the antisense strand comprises nucleotide sequencethat is complementary to a nucleotide sequence in a target nucleic acidmolecule or a portion thereof (i.e., an undesired gene) and the sensestrand comprises nucleotide sequence corresponding to the target nucleicacid sequence or a portion thereof. Alternatively, interfering RNA isassembled from a single oligonucleotide, where the self-complementarysense and antisense regions are linked by means of nucleic acid based ornon-nucleic acid-based linker(s). The interfering RNA can be apolynucleotide with a duplex, asymmetric duplex, hairpin or asymmetrichairpin secondary structure, having self-complementary sense andantisense regions, wherein the antisense region comprises a nucleotidesequence that is complementary to nucleotide sequence in a separatetarget nucleic acid molecule or a portion thereof and the sense regionhaving nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. The interfering can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active siRNA molecule capable of mediating RNA interference.

In some embodiments, the interfering RNA coding region encodes aself-complementary RNA molecule having a sense region, an antisenseregion and a loop region. Such an RNA molecule when expressed desirablyforms a “hairpin” structure, and is referred to herein as an “shRNA.”The loop region is generally between about 2 and about 10 nucleotides inlength. In some embodiments, the loop region is from about 6 to about 9nucleotides in length. In some embodiments, the sense region and theantisense region are between about 15 and about 20 nucleotides inlength. Following post-transcriptional processing, the small hairpin RNAis converted into a siRNA by a cleavage event mediated by the enzymeDicer, which is a member of the RNase III family. The siRNA is thencapable of inhibiting the expression of a gene with which it shareshomology. For details, see Brummelkamp et al., Science 296:550-553,(2002); Lee et al, Nature Biotechnol., 20, 500-505, (2002); Miyagishiand Taira, Nature Biotechnol 20:497-500, (2002); Paddison et al. Genes &Dev. 16:948-958, (2002); Paul,

Nature Biotechnol, 20, 505-508, (2002); Sui, Proc. Natl. Acad. Sd. USA,99(6), 5515-5520, (2002); Yu et al. Proc NatlAcadSci USA 99:6047-6052,(2002).

The target RNA cleavage reaction guided by siRNAs is highly sequencespecific. In general, siRNA containing a nucleotide sequences identicalto a portion of the target nucleic acid are preferred for inhibition.However, 100% sequence identity between the siRNA and the target gene isnot required to practice the present invention. Thus the invention hasthe advantage of being able to tolerate sequence variations that mightbe expected due to genetic mutation, strain polymorphism, orevolutionary divergence. For example, siRNA sequences with insertions,deletions, and single point mutations relative to the target sequencehave also been found to be effective for inhibition. Alternatively,siRNA sequences with nucleotide analog substitutions or insertions canbe effective for inhibition. In general the siRNAs must retainspecificity for their target, i.e., must not directly bind to, ordirectly significantly affect expression levels of, transcripts otherthan the intended target.

Rib ozymes

In some embodiments, the inhibitory nucleic acids are ribozymes.Trans-cleaving enzymatic nucleic acid molecules can also be used; theyhave shown promise as therapeutic agents for human disease (Usman &McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen andMarr, 1995 J. Med. Chem. 38, 2023-2037). Enzymatic nucleic acidmolecules can be designed to cleave specific lncRNA targets within thebackground of cellular RNA. Such a cleavage event renders the lncRNAnon-functional.

In general, enzymatic nucleic acids with RNA cleaving activity act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of a enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

Several approaches such as in vitro selection (evolution) strategies(Orgel, 1979, Proc. R. Soc. London, B 205, 435) have been used to evolvenew nucleic acid catalysts capable of catalyzing a variety of reactions,such as cleavage and ligation of phosphodiester linkages and amidelinkages, (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker etal, 1994, TIBTECH 12, 268; Bartel et al, 1993, Science 261 :1411-1418;Szostak, 1993, TIBS 17, 89-93; Kumar et al, 1995, FASEB J., 9, 1183;Breaker, 1996, Curr. Op. Biotech., 1, 442). The development of ribozymesthat are optimal for catalytic activity would contribute significantlyto any strategy that employs RNA-cleaving ribozymes for the purpose ofregulating gene expression. The hammerhead ribozyme, for example,functions with a catalytic rate (kcat) of about 1 min in the presence ofsaturating (10 MM) concentrations of Mg²⁺ cofactor. An artificial “RNAligase” ribozyme has been shown to catalyze the correspondingself-modification reaction with a rate of about 100 min⁻¹. In addition,it is known that certain modified hammerhead ribozymes that havesubstrate binding arms made of DNA catalyze RNA cleavage with multipleturn-over rates that approach 100 min⁻¹.

Making and Using Inhibitory Nucleic Acids

The nucleic acid sequences used to practice the methods describedherein, whether RNA, cDNA, genomic DNA, vectors, viruses or hybridsthereof, can be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly. Ifdesired, nucleic acid sequences of the invention can be inserted intodelivery vectors and expressed from transcription units within thevectors. The recombinant vectors can be DNA plasmids or viral vectors.Generation of the vector construct can be accomplished using anysuitable genetic engineering techniques well known in the art,including, without limitation, the standard techniques of PCR,oligonucleotide synthesis, restriction endonuclease digestion, ligation,transformation, plasmid purification, and DNA sequencing, for example asdescribed in Sambrook et al. Molecular Cloning: A Laboratory Manual.(1989)), Coffin et al. (Retroviruses. (1997)) and “RNA Viruses: APractical Approach” (Alan J. Cann, Ed., Oxford University Press,(2000)).

Preferably, inhibitory nucleic acids of the invention are synthesizedchemically. Nucleic acid sequences used to practice this invention canbe synthesized in vitro by well-known chemical synthesis techniques, asdescribed in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov(1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol.Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang(1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109;Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066;WO/2008/043753 and WO/2008/049085, and the refences cited therein.

Nucleic acid sequences of the invention can be stabilized againstnucleolytic degradation such as by the incorporation of a modification,e.g., a nucleotide modification. For example, nucleic acid sequences ofthe invention includes a phosphorothioate at least the first, second, orthird internucleotide linkage at the 5′ or 3′ end of the nucleotidesequence. As another example, the nucleic acid sequence can include a2′-modified nucleotide, e.g., a 2′-deoxy, 2′-deoxy-2′-fluoro,2′-O-methyl, 2′-O-ethoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O—NMA). As another example, the nucleic acidsequence can include at least one 2′-O-methyl-modified nucleotide, andin some embodiments, all of the nucleotides include a 2′-O-methylmodification. In some embodiments, the nucleic acids are “locked,” i.e.,comprise nucleic acid analogues in which the ribose ring is “locked” bya methylene bridge connecting the 2′-O atom and the 4′-C atom (see,e.g., Kaupinnen et al., Drug Disc. Today 2(3):287-290 (2005); Koshkin etal., J. Am. Chem. Soc., 120(50):13252-13253 (1998)). For additionalmodifications see US 20100004320, US 20090298916, and US 20090143326.

It is understood that any of the modified chemistries or formats ofinhibitory nucleic acids described herein can be combined with eachother, and that one, two, three, four, five, or more different types ofmodifications can be included within the same molecule.

Techniques for the manipulation of nucleic acids used to practice thisinvention, such as, e.g., subcloning, labeling probes (e.g.,random-primer labeling using Klenow polymerase, nick translation,amplification), sequencing, hybridization and the like are welldescribed in the scientific and patent literature, see, e.g., Sambrooket al., Molecular Cloning; A Laboratory Manual 3d ed. (2001); CurrentProtocols in Molecular Biology, Ausubel et al., eds. (John Wiley & Sons,Inc., New York 2010); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); Laboratory Techniques In Biochemistry AndMolecular Biology: Hybridization With Nucleic Acid Probes, Part I.Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Pharmaceutical Compositions

The methods described herein can include the administration ofpharmaceutical compositions and formulations comprising inhibitorynucleic acid sequences designed to target an lncRNA.

In some embodiments, the compositions are formulated with apharmaceutically acceptable carrier. The pharmaceutical compositions andformulations can be administered parenterally, topically, orally or bylocal administration, such as by aerosol or transdermally. Thepharmaceutical compositions can be formulated in any way and can beadministered in a variety of unit dosage forms depending upon thecondition or disease and the degree of illness, the general medicalcondition of each patient, the resulting preferred method ofadministration and the like. Details on techniques for formulation andadministration of pharmaceuticals are well described in the scientificand patent literature, see, e.g., Remington: The Science and Practice ofPharmacy, 21st ed., 2005.

The inhibitory nucleic acids can be administered alone or as a componentof a pharmaceutical formulation (composition). The compounds may beformulated for administration, in any convenient way for use in human orveterinary medicine. Wetting agents, emulsifiers and lubricants, such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, release agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe compositions.

Formulations of the compositions of the invention include those suitablefor intradermal, inhalation, oral/nasal, topical, parenteral, rectal,and/or intravaginal administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient (e.g.,nucleic acid sequences of this invention) which can be combined with acarrier material to produce a single dosage form will vary dependingupon the host being treated, the particular mode of administration,e.g., intradermal or inhalation. The amount of active ingredient whichcan be combined with a carrier material to produce a single dosage formwill generally be that amount of the compound which produces atherapeutic effect, e.g., an antigen specific T cell or humoralresponse.

Pharmaceutical formulations of this invention can be prepared accordingto any method known to the art for the manufacture of pharmaceuticals.Such drugs can contain sweetening agents, flavoring agents, coloringagents and preserving agents. A formulation can be admixtured withnontoxic pharmaceutically acceptable excipients which are suitable formanufacture. Formulations may comprise one or more diluents,emulsifiers, preservatives, buffers, excipients, etc. and may beprovided in such forms as liquids, powders, emulsions, lyophilizedpowders, sprays, creams, lotions, controlled release formulations,tablets, pills, gels, on patches, in implants, etc.

Pharmaceutical formulations for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art inappropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Pharmaceuticalpreparations for oral use can be formulated as a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragee cores. Suitable solid excipients arecarbohydrate or protein fillers include, e.g., sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such asthe cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate. Push-fit capsules can contain activeagents mixed with a filler or binders such as lactose or starches,lubricants such as talc or magnesium stearate, and, optionally,stabilizers. In soft capsules, the active agents can be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycol with or without stabilizers.

Aqueous suspensions can contain an active agent (e.g., nucleic acidsequences of the invention) in admixture with excipients suitable forthe manufacture of aqueous suspensions, e.g., for aqueous intradermalinjections. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethylene oxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). Theaqueous suspension can also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

In some embodiments, oil-based pharmaceuticals are used foradministration of nucleic acid sequences of the invention. Oil-basedsuspensions can be formulated by suspending an active agent in avegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin; or a mixture of these.See e.g., U.S. Pat. No. 5,716,928 describing using essential oils oressential oil components for increasing bioavailability and reducinginter- and intra-individual variability of orally administeredhydrophobic pharmaceutical compounds (see also U.S. Pat. No. 5,858,401).The oil suspensions can contain a thickening agent, such as beeswax,hard paraffin or cetyl alcohol. Sweetening agents can be added toprovide a palatable oral preparation, such as glycerol, sorbitol orsucrose. These formulations can be preserved by the addition of anantioxidant such as ascorbic acid. As an example of an injectable oilvehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.

Pharmaceutical formulations can also be in the form of oil-in-wateremulsions. The oily phase can be a vegetable oil or a mineral oil,described above, or a mixture of these. Suitable emulsifying agentsinclude naturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan mono-oleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. Theemulsion can also contain sweetening agents and flavoring agents, as inthe formulation of syrups and elixirs. Such formulations can alsocontain a demulcent, a preservative, or a coloring agent. In alternativeembodiments, these injectable oil-in-water emulsions of the inventioncomprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitanmonooleate and/or an ethoxylated sorbitan trioleate.

The pharmaceutical compounds can also be administered by in intranasal,intraocular and intravaginal routes including suppositories,insufflation, powders and aerosol formulations (for examples of steroidinhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193;Tjwa (1995) Ann. Allergy Asthma Immunol. 75:107-111).

Suppositories formulations can be prepared by mixing the drug with asuitable non-irritating excipient which is solid at ordinarytemperatures but liquid at body temperatures and will therefore melt inthe body to release the drug. Such materials are cocoa butter andpolyethylene glycols.

In some embodiments, the pharmaceutical compounds can be deliveredtransdermally, by a topical route, formulated as applicator sticks,solutions, suspensions, emulsions, gels, creams, ointments, pastes,jellies, paints, powders, and aerosols.

In some embodiments, the pharmaceutical compounds can also be deliveredas microspheres for slow release in the body. For example, microspherescan be administered via intradermal injection of drug which slowlyrelease subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed.7:623-645; as biodegradable and injectable gel formulations, see, e.g.,Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oraladministration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.

In some embodiments, the pharmaceutical compounds can be parenterallyadministered, such as by intravenous (IV) administration oradministration into a body cavity or lumen of an organ. Theseformulations can comprise a solution of active agent dissolved in apharmaceutically acceptable carrier. Acceptable vehicles and solventsthat can be employed are water and Ringer's solution, an isotonic sodiumchloride. In addition, sterile fixed oils can be employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid can likewise be used in the preparation ofinjectables. These solutions are sterile and generally free ofundesirable matter. These formulations may be sterilized byconventional, well known sterilization techniques. The formulations maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents, e.g., sodium acetate, sodiumchloride, potassium chloride, calcium chloride, sodium lactate and thelike. The concentration of active agent in these formulations can varywidely, and will be selected primarily based on fluid volumes,viscosities, body weight, and the like, in accordance with theparticular mode of administration selected and the patient's needs. ForIV administration, the formulation can be a sterile injectablepreparation, such as a sterile injectable aqueous or oleaginoussuspension. This suspension can be formulated using those suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol. The administration can be by bolus or continuousinfusion (e.g., substantially uninterrupted introduction into a bloodvessel for a specified period of time).

In some embodiments, the pharmaceutical compounds and formulations canbe lyophilized. Stable lyophilized formulations comprising an inhibitorynucleic acid can be made by lyophilizing a solution comprising apharmaceutical of the invention and a bulking agent, e.g., mannitol,trehalose, raffinose, and sucrose or mixtures thereof. A process forpreparing a stable lyophilized formulation can include lyophilizing asolution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mLNaCl, and a sodium citrate buffer having a pH greater than 5.5 but lessthan 6.5. See, e.g., U.S. 20040028670.

The compositions and formulations can be delivered by the use ofliposomes. By using liposomes, particularly where the liposome surfacecarries ligands specific for target cells, or are otherwisepreferentially directed to a specific organ, one can focus the deliveryof the active agent into target cells in vivo. See, e.g., U.S. Pat. Nos.6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306;Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J.Hosp. Pharm. 46:1576-1587. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in abilayer or bilayers. Liposomes are unilamellar or multilamellar vesiclesthat have a membrane formed from a lipophilic material and an aqueousinterior that contains the composition to be delivered. Cationicliposomes are positively charged liposomes that are believed to interactwith negatively charged DNA molecules to form a stable complex.Liposomes that are pH-sensitive or negatively-charged are believed toentrap DNA rather than complex with it. Both cationic and noncationicliposomes have been used to deliver DNA to cells.

Liposomes can also include “sterically stabilized” liposomes, i.e.,liposomes comprising one or more specialized lipids. When incorporatedinto liposomes, these specialized lipids result in liposomes withenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposomecomprises one or more glycolipids or is derivatized with one or morehydrophilic polymers, such as a polyethylene glycol (PEG) moiety.Liposomes and their uses are further described in U.S. Pat. No.6,287,860.

The formulations of the invention can be administered for prophylacticand/or therapeutic treatments. In some embodiments, for therapeuticapplications, compositions are administered to a subject who is need ofreduced triglyceride levels, or who is at risk of or has a disorderdescribed herein, in an amount sufficient to cure, alleviate orpartially arrest the clinical manifestations of the disorder or itscomplications; this can be called a therapeutically effective amount.For example, in some embodiments, pharmaceutical compositions of theinvention are administered in an amount sufficient to decrease serumlevels of triglycerides in the subject.

The amount of pharmaceutical composition adequate to accomplish this isa therapeutically effective dose. The dosage schedule and amountseffective for this use, i.e., the dosing regimen, will depend upon avariety of factors, including the stage of the disease or condition, theseverity of the disease or condition, the general state of the patient'shealth, the patient's physical status, age and the like. In calculatingthe dosage regimen for a patient, the mode of administration also istaken into consideration. The dosage regimen also takes intoconsideration pharmacokinetics parameters well known in the art, i.e.,the active agents' rate of absorption, bioavailability, metabolism,clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. SteroidBiochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341;Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci.84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur.J. Clin. Pharmacol. 24:103-108; Remington: The Science and Practice ofPharmacy, 21st ed., 2005). The state of the art allows the clinician todetermine the dosage regimen for each individual patient, active agentand disease or condition treated. Guidelines provided for similarcompositions used as pharmaceuticals can be used as guidance todetermine the dosage regiment, i.e., dose schedule and dosage levels,administered practicing the methods of the invention are correct andappropriate.

Single or multiple administrations of formulations can be givendepending on for example: the dosage and frequency as required andtolerated by the patient, the degree and amount of therapeutic effectgenerated after each administration (e.g., effect on tumor size orgrowth), and the like. The formulations should provide a sufficientquantity of active agent to effectively treat, prevent or ameliorateconditions, diseases or symptoms.

In alternative embodiments, pharmaceutical formulations for oraladministration are in a daily amount of between about 1 to 100 or moremg per kilogram of body weight per day. Lower dosages can be used, incontrast to administration orally, into the blood stream, into a bodycavity or into a lumen of an organ. Substantially higher dosages can beused in topical or oral administration or administering by powders,spray or inhalation. Actual methods for preparing parenterally ornon-parenterally administrable formulations will be known or apparent tothose skilled in the art and are described in more detail in suchpublications as Remington: The Science and Practice of Pharmacy, 21sted., 2005.

Various studies have reported successful mammalian dosing usingcomplementary nucleic acid sequences. For example, Esau C., et al.,(2006) Cell Metabolism, 3(2):87-98 reported dosing of normal mice withintraperitoneal doses of miR-122 antisense oligonucleotide ranging from12.5 to 75 mg/kg twice weekly for 4 weeks. The mice appeared healthy andnormal at the end of treatment, with no loss of body weight or reducedfood intake. Plasma transaminase levels were in the normal range (AST ¾45, ALT ¾ 35) for all doses with the exception of the 75 mg/kg dose ofmiR-122 ASO, which showed a very mild increase in ALT and AST levels.They concluded that 50mg/kg was an effective, non-toxic dose. Anotherstudy by Krutzfeldt

J., et al., (2005) Nature 438, 685-689, injected anatgomirs to silencemiR-122 in mice using a total dose of 80, 160 or 240 mg per kg bodyweight. The highest dose resulted in a complete loss of miR-122 signal.In yet another study, locked nucleic acid molecules (“LNA molecules”)were successfully applied in primates to silence miR-122. Elmen J., etal., (2008) Nature 452, 896-899, report that efficient silencing ofmiR-122 was achieved in primates by three doses of 10 mg kg-1LNA-antimiR, leading to a long-lasting and reversible decrease in totalplasma cholesterol without any evidence for LNA-associated toxicities orhistopathological changes in the study animals.

In some embodiments, the methods described herein can includeco-administration with other drugs or pharmaceuticals, e.g.,compositions for providing cholesterol homeostasis. For example, theinhibitory nucleic acids can be co-administered with drugs for treatingor reducing risk of a disorder described herein.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Materials and Methods

-   -   The following materials and methods were used in the Examples        1-7 set forth below.

RIP-Seq

RNA immunoprecipitation was performed (Zhao et al., 2008) using 10⁷wildtype 16.7 (Lee and Lu, 1999) and Ezh2−/− (Shen et al., 2008) EScells. To construct RIP-seq libraries, cell nuclei were isolated,nuclear lysates were prepared, treated with 400 U/ml DNAse, andincubated with anti-Ezh2 antibodies (Active Motif) or control IgG (CellSignaling Technology). RNA-protein complexes were immunoprecipitatedwith protein A agarose beads and RNA extracted using Trizol(Invitrogen). To preserve strand information, template switching wasused for the library construction (Cloonan et al., 2008). 20-150 ng RNAand Adaptorl (5′-CTTTCCCTACACGACGCTCTTCCGATCT -3′; SEQ ID NO: 193050)were used for first-strand cDNA synthesis using Superscript II ReverseTranscription Kit (Invitrogen). Superscript II adds non-template CCC 3′overhangs, which were used to hybridize to Adaptor2-GGG template-switchprimer (5′-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGGG-3′; SEQ ID NO:

193051). During 1^(st)-strand cDNA synthesis, samples were incubatedwith adaptorl at 20° C. for 10 min, followed by 37° C. for 10 min and42° C. for 45 min. Denatured template switch primer was then added andeach tube incubated for 30 min at 42° C., followed by 75° C. for 15 min.Resulting cDNAs were amplified by forward(5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′; SEQID NO: 193052) and reverse (5′-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3′;SEQ ID NO: 193053) Illumina primers. PCR was performed by Phusionpolymerase (BioRad) as follows: 98° C. for 30 s, 20-24 cycles of [98° C.10 s, 65° C. 30 s, 72° C. 30 s], and 72° C. for 5 min. PCR products wereloaded on 3% NuSieve gel for size-selection and 200-1,200 bp productswere excised and extracted by QIAEX II Agarose Gel Extraction Kit(Qiagen). Minus-RT samples generally yielded no products. DNAconcentrations were quantitated by PicoGreen. 5-10 ml of 2-20 nM cDNAsamples were sequenced by the Sequencing Core Facility of the Dept. ofMolecular Biology, MGH, on the Illumina GAIL

Bioinformatic Analysis

Complete RIP-seq datasets can be accessed through GEO via seriesGSE17064. Except as noted below, all analyses were performed usingcustom C++ programs. Image processing and base calling were performedusing the Illumina pipeline. 3′ adaptor sequences were detected bycrossmatch and matches of ≥5 bases were trimmed, homopolymer readsfiltered, and reads matching the mitochondrial genome and ribosomal RNAsexcluded from all subsequent analyses. Remaining sequences were thenaligned to the mm9 mouse reference genome using shortQueryLookup(Batzoglou et al., 2002). Alignments with ≤1 error were retained.Because library construction and sequencing generate sequence from theopposite strand of the PRC2-bound RNA, in all further analysis, wetreated each read as if it were reverse-complemented. To determine thecorrelation coefficients comparing the original a-Ezh2 RIP-seq libraryto its technical and biological replicates and also to RIP-seq of theEzh2−/− control line, we compared the number of reads per gene betweentwo samples and, for each pair, we computed the Pearson correlationbetween the number of reads mapped to each refGene. That is, for eachsample, we created a vector of counts of reads mapped to each refGeneand computed the Pearson correlation between all pairs of vectors.

Locations of repetitive sequences in mm9 (RepeatMasker) were obtainedfrom the UCSC Genome Browser database (Kent et al., The human genomebrowser at UCSC. Genome Res. 2002 June; 12(6):996-1006; Fujita et al.,“The UCSC Genome Browser database: update 2011.” Nucleic Acids Res. 2010Oct. 18) The overlap of PRC2 transcriptome reads with these repeats wasobtained by intersecting coordinates of RepeatMasker data withcoordinates of read alignments. The UCSC transcriptome was used asgeneral reference (available online athgdownload.cse.ucsc.edu/goldenPath/mm9/database/transcriptome.txt.gz).To obtain a set of non-overlapping distinct transcribed regions, wesorted the UCSC transcriptome transcripts by start coordinate and mergedoverlapping transcripts on the same strand (joined UCSC transcriptome:39,003 transcripts total). We then intersected read alignmentcoordinates with those of the merged UCSC transcripts to determine thenumber of UCSC transcripts present in the PRC2 transcriptome. Hits tothe transcripts were converted to RPKM units, where the read count is1/(n*K*M), and n is the number of alignments in the genome, K is thetranscript length divided by 1,000, and M is the sequencing depthincluding only reads mapping to mm9 divided by 1,000,000 (Mortazavi etal., 2008). This normalization allows for comparisons betweentranscripts of differing lengths and between samples of differingsequencing depths. To generate promoter maps, promoter regions weredefined as −10,000 to +2000 bases relative to TSS (obtained from refGenecatalog, UCSC Genome Browser,). We plotted read counts overlappingpromoter regions, except that the limit of 10 alignments was relaxed.For the chromosomal alignments in FIG. 1H and FIGS. 7-12, read numberswere computed for all non-overlapping consecutive 100 kb windows on eachchromosome. Reads were normalized such that those mapping to n locationswere counted as 1/n^(th) of a read at each location. Graphs were plottedusing custom scripts written in R. To generate Tables 3-7, a list of allenriched transcripts were found by comparing the RPKM scores on eachstrand for all transcripts in the WT and Ezh2−/− samples. Then theircoordinates were intersected with coordinates of the feature ofinterest. Features not in NCBI37/mm9 mouse assembly coordinates wereconverted to those coordinates using UCSC's LiftOver utility (TheliftOver utility effectively maps one genome to another, allowing rapididentification of regions of interest between successive assemblies ofthe same species or between two distinct species; available online atgenome.ucsc.edu/cgi-bin/hgLiftOver). Only features whose coordinateswere convertible are shown.

RIP/qRT-PCR

Validation RIPs were performed as described (Zhao et al., 2008) using 5ul of rabbit anti-mouse-Ezh2 antibodies (Active Motif) or normal rabbitIgG (Millipore). RIP was followed by quantitative, strand-specificRT-PCR using the ICYCLER IQ™ Real-time detection system (BioRad).Gene-specific PCR primer pairs are:

Malat-1: Forward SEQ ID NO: 193054 5′-GCCTTTTGTCACCTCACT-3′; ReverseSEQ ID NO: 193055 5′-CAAACTCACTGCAAGGTCTC-3′; Malatl-as: ForwardSEQ ID NO: 193056 5′-TACTGGGTCTGGATTCTCTG-3′; Reverse SEQ ID NO: 1930575′-CAGTTCCGTGGTCTTTAGTG-3′; Foxn2-as: Forward SEQ ID NO: 1930585′-GGCTATGCTCATGCTGTAAC; Reverse SEQ ID NO: 1930595′-GTTACTGGCATCTTTCTCACA-3′; Ly6e-as: Forward SEQ ID NO: 1930605′-CCACACCGAGATTGAGATTG-3′; Reverse SEQ ID NO: 1930615′-GCCAGGAGAAAGACCATTAC-3′; Bgn-as: Forward SEQ ID NO: 1930625′-TGTGAACCCTTTCCTGGA-3′; Reverse SEQ ID NO: 1930635′-CTTCACAGGTCTCTAGCCA-3′; Gtl2: Forward SEQ ID NO: 1930645′-CGAGGACTTCACGCACAAC -3′; Reverse SEQ ID NO: 1930655′-TTACAGTTGGAGGGTCCTGG-3′; Gtl2-as: Forward SEQ ID NO: 1930665′-CACCCTGAACATCCAACA-3′; Reverse SEQ ID NO: 1930675′-CATCTGCTTTTCCTACCTGG-3′; Hapal-upstream: Forward SEQ ID NO: 1930685′-GGTCCAAAATCGGCAGT-3′; Reverse SEQ ID NO: 1930695′-GTCCTCAAATCCCTACCAGA-3′; Htr6-downstream: Forward SEQ ID NO: 1930705′-ACACGGTCGTGAAGCTAGGTA-3′;  Reverse SEQ ID NO: 1930715′-CAGTTGGAGTAGGCCATTCCC-3′; Nespas/TR019501: Forward SEQ ID NO: 1930725′-AGATGAGTCCAGGTGCTT-3′; Reverse SEQ ID NO: 1930735′-CAAGTCCAGAGTAGCCAAC-3′;

Xist-Forward 3F5 and -Reverse 2R primers have been described (Zhao etal., 2008). For strand-specific cDNA synthesis, the reverse primer wasused, qPCR carried out with SYBR green (BioRad), and threshold crossings(Ct) recorded. Each value was normalized to input RNA levels.

Northern Blot Analysis

5 μg of poly(A+) RNA were isolated from 16.7 ES cells, separated by 0.8%agarose gel containing formaldehyde, blotted onto Hybond-XL (GEHealthcare), and hybridized to probe using Ultrahyb (Ambion) at 42° C.Probes were generated using STRIP-EZ PCR kit (Ambion) and amplified fromgenomic DNA with:

Malat1-AS-F, SEQ ID NO: 193074 5′-TGGGCTATTTTTCCTTACTGG-3′; Malat1-AS-R,SEQ ID NO: 193075 5′-GAGTCCCTTTGCTGTGCTG-3′; (Gtl2) Meg3-F,SEQ ID NO: 193076 5′-GCGATAAAGGAAGACACATGC-3′; Meg3-R, SEQ ID NO: 1930775′-CCACTCCTTACTGGCTGCTC-3′; Meg3 ds-F3, SEQ ID NO: 1930785′-ATGAAGTCCATGGTGACAGAC-3′; Meg3 ds-R2, SEQ ID NO: 1930795′-ACGCTCTCGCATACACAATG-3′; Rtl1-F, SEQ ID NO: 1930805′-GTTGGGGATGAAGATGTCGT-3′; Rtl1-R, SEQ ID NO: 1930815′-GAGGCACAAGGGAAAATGAC-3′;  Nespas ds-F, SEQ ID NO: 1930825′-TGGACTTGCTACCCAAAAGG-3′; Nespas ds-R, SEQ ID NO: 1930835′-CGATGTTGCCCAGTTATCAG-3′; Bgn-AS-F, SEQ ID NO: 1930845′-CAACTGACCTCATAAGCAGCAC-3′; Bgn-AS-R, SEQ ID NO: 1930855′-AGGCTGCTTTCTGCTTCACA-3′; Htr6 up-F, SEQ ID NO: 1930865′-ATACTGAAGTGCCCGGAGTG-3′; Htr6 up-R, SEQ ID NO: 1930875′-CAGGGGACAGACATCAGTGAG-3′;.

UV-Crosslink RIP

UV-crosslink IP was performed as described (Ule et al., 2005), exceptthat transcripts in the RNA-protein complexes were not trimmed by RNAsetreatment prior to RNA isolation in order to preserve full-length RNAfor RT-PCR. Mouse ES cells were UV-irradiated at 254 nm, 400 mJ/cm²(using a Stratagene STRATALINKER), cell nuclei were lysed in RSB-TRITONbuffer (10 mM Tris-HCl, 100 mM NaCl, 2.5 mM MgCl₂, 35 μg/mL digitonin,0.5% triton X-100) with disruptive sonication. Nuclear lysates werepre-cleared with salmon sperm DNA/protein agarose beads for 1 hr at 4°C. and incubated with antibodies overnight. RNA/antibody complexes werethen precipitated with Protein A DYNABEADS (Invitrogen), washed first ina low-stringency buffer (1XPBS [150 mM NaCl], 0.1% SDS, 0.5%deoxycholate, 0.5% NP-40), then washed twice in a high-stringency,high-salt buffer (SXPBS [750 mM NaCl], 0.1% SDS, 0.5% deoxycholate, 0.5%NP-40), and treated with proteinase K. RNA was extracted using TRIZOL(Invitrogen) and RT-qPCR was performed as described above.

Expression and Purification of Human PRC2 Components

For expression of human PRC2 subunits, N-terminal flagged-tagged EZH2and SUZ12 in pFastBacl were expressed in Sf9 cells (Francis et al.,2001). For expression of the whole PRC2 complex, flag-tagged EZH2 wascoexpressed with untagged SUZ12, EED, and RBAP48. Extracts were made byfour freeze-thaw cycles in BC300 buffer (20 mM HEPES pH 7.9, 300 mM KCl,0.2 mM EDTA, 10% glycerol, 1 mM DTT, 0.2 mM PMSF, and complete proteaseinhibitors (Roche)) and bound to M2 beads for 4 h and washed with BC2000before eluting in BC300 with 0.4 mg/ml flag peptide. EZH2 and PRC2 wereadjusted to 100 mM KCl and loaded onto a HiTrap Heparin FF lml columnand eluted with a 100-1000 mM KCl gradient. Peak fractions wereconcentrated using Amicon ultra 10 kDa MWCO concentrators (Millipore)and loaded onto a Superose 6 column equilibrated with BC300. Peakfractions were collected and concentrated. For SUZ12, the flag elutionwas concentrated and loaded onto a Superdex 200 column equilibrated withBC300.

Electrophoretic Mobility Shifting Assays (EMSA)

RNA-EMSA is performed as previously described (Zhao et al., 2008). The30 nt Hes-1 probe (-270 bp downstream of TSS in an antisense direction)was used for gel shifts. RNA probes were radiolabeled with [γ-33p]ATPusing T4 polynucleotide kinase (Ambion). Purified PRC2 proteins (1 μg)were incubated with labeled probe for 1 hr at 4 C. RNA-protein complexeswere separated on a 4% non-denaturing polyacrylamide gel in 0.5×TBE at250 V at 4° C. for 1 h. Gels were dried and exposed to Kodak BioMaxfilm.

RNA Pulldown Assays

We incorporated T7 promoter sequence into forward primers for PCRproducts of RepA, Xist exon 1, and truncated Gtl2. Full-length Gtl2 wascloned into pYX-ASC and XistE1 into pEF1/V5/HisB (Invitrogen). Specificprimer sequences were:

RepA-F: SEQ ID NO: 193088TAATACGACTCACTATAGGGAGAcccatcggggccacggatacctgtgtgt cc; RepA-R: SEQ ID NO: 193089 taataggtgaggtttcaatgatttacatcg;  Truncated-Gtl2-F:SEQ ID NO: 193090 TAATACGACTCACTATAGGGAGATTCTGAGACACTGACCATGTGCCCAGTGCACC; Truncated-Gtl2-R: SEQ ID NO: 193091CGTCGTGGGTGGAGTCCTCGCGCTGGGCTTCC; Xist E1-F: SEQ ID NO: 193092atgctctgtgtcctctatcaga; Xist E1-R: SEQ ID NO: 193093gaagtcagtatggagggggt;

RNAs were then transcribed using the Mega Script T7 (Ambion), purifiedusing Trizol, and slow-cooled to facilitate secondary structureformation. For pulldown assays, 3 μg of Flag-PRC2 or Flag-GFP and 5 pmolof RNA supplemented with 20U RNAsin were incubated for 30 min on ice. 10μl of flag beads were added and incubated on a rotating wheel at 4° C.for 1 hr. Beads were washed 3 times with 200 μl buffer containing 150mMKCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT, 0.5% NP40 and 1 mM PMSF.RNA-protein complexes were eluted from flag beads by addition of 35 μlof 0.2M-glycine pH2.5. Eluates were neutralized by addition of 1/10^(th)volume of 1 M Tris pH 8.0 and analyzed by gel electrophoresis.

Knockdown Analysis and qRT-PCR

shRNA oligos were cloned into MISSION pLKO. 1-puro (Sigma-Aldrich)vector and transfected into wild-type mouse ES cells by Lipofectamine2000 (Invitrogen). After 10 days of puromycin selection, cells werecollected and qRT-PCR was performed to confirm RNA knockdown. Thecorresponding scrambled sequence (MISSION Non-target shRNA) was used asa control (Scr). The shRNA oligos for Gtl2: (Top strand) 5′-CCG GGC AAGTGA GAG GAC ACA TAG GCT CGA GCC TAT GTG TCC TCTCAC TTG CTT TTT G-3′; SEQID NO: 193094 (Bottom strand) 5′-AAT TCA AAA AGC AAG TGA GAG GAC ACA TAGGCT CGA GCC TAT GTG TCC TCT CACTTG C -3; SEQ ID NO: 193095. qPCR primersfor Gtl2 and Gtl2-as RNAs are as described above. Primers for Dlk1 RNAs:(Forward) 5′-ACG GGA AAT TCT GCG AAA TA-3′; SEQ ID NO: 193096 (Reverse)5′-CTT TCC AGA GAA CCC AGG TG-3′; SEQ ID NO: 193097. Another Gtl2 shRNAwas purchased from Open Biosystems (V2MM_97929). Ezh2 levels afterknockdown with this shRNA were tested by qPCR (Zhao et al., 2008). Aftertesting multiple clones, we concluded that Gtl2 could be knocked down inearly passage clones (50-70%), but knockdown clones were difficult tomaintain in culture long-term.

DNA ChIP and Real-Time PCR

ChIP was performed as described (Zhao et al., 2008). 5 μl of α-Ezh2antibodies (Active Motif 39103), normal rabbit IgG (Upstate 12-370), andα-H3K27me3 (Upstate) were used per IP. Real-time PCR for ChIP DNA wasperformed at the Gtl2-proximal DMR with prGtl2F/prGtl2R, at theGtl2-distal DMR with DMR-F/DMR-R, at the Dlk1 promoter withprDlk1F/prDlk1R, and at the Gapdh promoter with prGAPDH-F/prGAPDH-R.Primer sequences are as follows:

proximal-DMR SEQ ID NO: 193098 5′-CATTACCACAGGGACCCCATTTT; proximal-DMRSEQ ID NO: 193099 5′-GATACGGGGAATTTGGCATTGTT; prDlk1F SEQ ID NO: 1931005′-CTGTCTGCATTTGACGGTGAAC; prDlk1R SEQ ID NO: 1931015′-CTCCTCTCGCAGGTACCACAGT; distal-DMR-F SEQ ID NO: 1931025′- GCCGTAAAGATGACCACA; distal-DMR-R SEQ ID NO: 1931035′- GGAGAAACCCCTAAGCTGTA; prGAPDH-F SEQ ID NO: 1931045′-AGCATCCCTAGACCCGTACAGT; prGAPDH-R SEQ ID NO: 1931055′-GGGTTCCTATAAATACGGACTGC; prActin-F SEQ ID NO: 1931065′-GCA GGC CTA GTA ACC GAG ACA; prActin-R SEQ ID NO: 1931075′-AGT TTT GGC GAT GGG TGC T;

The following materials and methods were used in Examples 10-15 setforth below.

LNA Nucleofection—2×10⁶ SV40T transformed MEFs were resuspended in 100μl of Mef nucleofector solution (Lonza). Cy3-labeled LNA molecules wereadded to a final concentration of 2 μM. The cells were transfected usingthe T-20 program. 2 ml of culture medium was added to the cells and 100μl of this suspension was plated on one gelatinized 10 well slide pertimepoint. LNA sequences were designed using Exiqon software (availableat exiqon.com). Modified LNA bases were strategically introduced tomaximize target affinity (Tm) while minimizing self-hybridization score.The LNA molecule sequences (from 5′ to 3′) were as follows:

LNA-Scr, SEQ ID NO: 193108 GTGTAACACGTCTATACGCCCA; LNA-C1,SEQ ID NO: 193109 CACTGCATTTTAGCA; LNA-C2, SEQ ID NO: 193110AAGTCAGTATGGAG; LNA-B, SEQ ID NO: 193111 AGGGGCTGGGGCTGG; LNA-E,SEQ ID NO: 193112 ATAGACACACAAAGCA; LNA-F, SEQ ID NO: 193113AAAGCCCGCCAA; LNA-4978, SEQ ID NO: 193114 GCTAAATGCACACAGGG; LNA-5205,SEQ ID NO: 193115 CAGTGCAGAGGTTTTT; LNA-726, SEQ ID NO: 193116TGCAATAACTCACAAAACCA; LNA-3′, SEQ ID NO: 193117 ACCCACCCATCCACCCACCC;

Real Time PCR—Total RNA was extracted after nucleofection using Trizol(Invitrogen). Reverse transcriptase reaction was performed using theSuperscript II kit and real time PCR performed on cDNA samples usingicycler SYBR green chemistry (Biorad).

ChIP—Cells were fixed at various time points after nucleofection in 1%formaldehyde solution. Fixation was stopped by addition of glycine to0.125M and ChIP was performed as described earlier (28) and quantitatedby qPCR.

Antibodies—The antibodies for various epitopes were purchased asfollows: H3K27me3, Active Motif 39535. Ezh2, Active Motif 39639 and BDPharmingen 612666. For Immunostaining, H3K27me3 antibodies were used at1:100 dilution and Ezh2 antibodies (BD Pharmingen) at 1:500. Alexa-Fluorsecondary antibodies were from Invitrogen. For Western blots, Ezh2antibodies (BD Pharmingen) were used at 1:2000 dilution. Actin antibody(Sigma A2066) was used at 1:5000 dilution.

DNA FISH, RNA FISH, and Immunostaining—Cells were grown on gelatinizedglass slides or cytospun. RNA FISH, DNA FISH, serial RNA-DNA FISH,immunostaining, and immunoFlSH were performed as described (24). XistRNA FISH was performed using nick-translated pSx9-3 probe or an Xistriboprobe cocktail. pSx9-3 was used as probe for Xist DNA FISH. Formetaphase spreads, colchicine was added to cells for 1 hr. Cells weretrypsinized and resuspended in 3 ml of 0.056M KCl for 30 minutes at roomtemperature, centrifuged and resuspended in methanol:acetic acid (3:1)fixative. After several changes of fixative, cells were dropped on achilled slide and processed for RNA or DNA FISH.

Example 1 Capturing the PRC2 Transcriptome by RIP-Seq

Native RNA immunoprecipitations (RIP) previously identified RepA, Xist,and Tsix as PRC2-interacting RNAs (Zhao et al., 2008). Here, wedeveloped a method of capturing the genome-wide pool bound to PRC2 bycombining native RIP (Zhao et al., 2008) and RNA-seq (Cloonan et al.,2008) (this method is referred to herein as “RIP-seq;” see an exemplaryFIG. 1A). Nuclear RNAs immunoprecipitated by α-Ezh2 antibodies wereisolated from mouse ES cells (Lee and Lu, 1999) and an Ezh2−/− control(Shen et al., 2008) (FIG. 1B), cDNAs created using strand-specificadaptors, and those from 200-1,200 nt were purified and subjected toIllumina sequencing (FIG. 1C).

In pilot experiments, we performed RIP on 10⁷ ES cells and includedseveral control RIPs to assess the specificity of α-Ezh2 pulldowns. Inthe wildtype pulldown and its technical and biological replicates,α-Ezh2 antibodies precipitated 70-170 ng of RNA from 107 ES cells andyielded a cDNA smear of >200 nt (FIG. 1C, FIG. 7A). Treatment withRNAses eliminated products in this size range (FIG. 7B) and —RT samplesyielded no products, suggesting that the immunoprecipitated material wasindeed RNA. There was ˜10-fold less RNA in the Ezh2−/− pulldown (˜14 ng)and when wildtype cells were immunoprecipitated by IgG (−24 ng). A500-fold enrichment over a mock RIP control (no cells) was alsoobserved. In the >200 nt size range, control RIPs (null cells, IgGpulldowns, mock) were even further depleted of RNA, as these sampleswere dominated by adaptor and primer dimers. We computationally filteredout adaptor/primer dimers, rRNA, mitochondrial RNA, reads with <18 nt orindeterminate nucleotides, and homopolymer runs in excess of 15 bases(FIGS. 7A-C). From an equivalent number of cells, control RIPs weresignificantly depleted of reads (FIG. 1D). In wildtype libraries,231,880-1.2 million reads remained after filtering. By contrast, only4,888 to 73,691 reads remained in controls (FIG. 1D, columns 2 and 3).The overwhelming majority of transcripts in the controls were ofspurious nature (adaptor/primer dimers, homopolymers, etc.). Therefore,wildtype RIPs exhibited substantial RNA enrichment and greater degreesof RNA complexity in comparison to control RIPs.

Approximately half of all reads in the wildtype libraries wasrepresented three times or more. Even after removing duplicates to avoidpotential PCR artifacts, the wildtype library contained 301,427 distinctreads (technical and biological replicates with 98,704 and 87,128,respectively), whereas control samples yielded only 1,050 (IgG) and17,424 (null) (FIG. 1D). The wildtype libraries were highly similaramong each other, with correlation coefficients (CC) of 0.71-0.90, ascompared to 0.27-0.01 when compared against Ezh2−/− and IgG controls,respectively (FIG. 1E). Reads mapping to repetitive elements of >10copies/genome accounted for <20% of total wildtype reads (FIG. 1F), withsimple repeats being most common and accounting for 85.714%, whereasLINEs, SINEs, and LTRs were relatively under-represented (FIG. 1G).Because reads with ≤10 alignments have greatest representation, wehereafter focus analysis on these reads (a cutoff of ≤10 retains geneswith low-copy genomic duplications).

We next examined their genome distribution by plotting distinct reads asa function of chromosome position (FIGS. 8-12). The alignments showedthat PRC2-associated RNAs occurred on every chromosome in the wildtypelibraries. Alignments for IgG and Ezh2−/− controls demonstrated few andsporadic reads. Therefore, our RIP-seq produced a specific andreproducible profile for the PRC2 transcriptome. A large number ofwildtype reads hits the X-chromosome (FIG. 1H), and a zoom of theX-inactivation center showed that our positive controls—Tsix, RepA, andXist RNAs—were each represented dozens of times (FIG. 11). The highsensitivity of our RIP-seq detection was suggested by representation ofRepA and Xist, which are in aggregate expressed at <10 copies/ES cell(Zhao et al., 2008). On the other hand, no hits occurred within othernoncoding RNAs of the X-inactivation center. Thus, the RIP-seq techniquewas both sensitive and specific.

Example 2 The PRC2 Transcriptome

To obtain saturating coverage, we scaled up sequencing and obtained 31.9million reads for the original wildtype sample and 36.4 million for itsbiological replicate. After removing duplicates and filtering as shownin FIG. 7A, U.S. Pat. Nos. 1,030,708 and 852,635 distinct reads ofalignment ≤10 remained for each library, respectively. These reads werethen combined with pilot wildtype reads for subsequent analyses(henceforth, WT library) and all analyses were performed using theEzh2−/− library as control.

To determine a threshold for calling transcripts a member of the “PRC2transcriptome”, we designed a strategy based on (i) the number ofdistinct reads per transcript, on the principle that bona fidePRC2-interacting transcripts should have higher read densities thanbackground, and (ii) the relative representation in the WT versus nulllibraries, reasoning that bona fide positives should be enriched in theWT. We calculated genic representations using “reads per kilobase permillion reads” (RPKM) as a means of normalizing for gene length anddepth of sequencing (Mortazavi et al., 2008), and then mapped all 39,003transcripts in the UCSC joined transcriptome to a scatterplot by their

WT RPKM (x-axis) and their null RPKM (y-axis) values (FIG. 2A).Transcripts with zero or near-zero representation in both librariesaccounted for the vast majority of datapoints [blue cloud at (0,0)].Transcripts with nonzero x-values and a zero y-value indicated apopulation represented only in WT pulldowns (FIG. 2A, y=0 line). Weestablished a density minimum by using control transcripts ascalibration points. Xist/RepA scored an RPKM of 4.19, implying 126distinct reads per million. Tsix scored 10.35, and Bsn-pasr (˜300-ntBsn-promoter associated transcript (Kanhere et al., 2010)) scored 0.95.The imprinted antisense transcript, Kcnq1ot1, has been proposed tointeract with PRC2, though whether it does so directly is not known(Pandey et al., 2008). Kcnq1ot1 scored 1.17. For negative controls, weused transcripts that a priori should not be in the WT library. Forexample, Hotair is expressed later in development only in caudal tissues(Rinn et al., 2007). It scored 0.25, implying only a singlerepresentation per million. Two other promoter-associated RNAs,Hey1-pasr and Pax3-pasr (Kanhere et al., 2010), are <200 nt and felloutside of our size-selection scheme. They scored 0.28 and 0.11,respectively, suggesting <<1 distinct reads per million. Cytoplasmicallylocalized protein-coding mRNAs that are not expected to bePRC2-interacting also showed low RPKM [Ins16 0.27, Ccdc8 0.22]. Weconsider these low representations background. On the basis of thecalibration points, we set the RPKM minimum at x=0.40, which fallsbetween values for positive and negative controls.

To determine an appropriate enrichment threshold, we examined WT/nullRPKM ratios for the same calibrators. Xist/RepA scored 4.18/0, implyinghundreds to thousands of representations in the WT library but none inthe null. Tsix scored 10.35/3.27, Bsn-pasr 0.95/0, and Kcnq1ot1 1.17/0.The negative controls scored low ratios, with Pax3-pasr at 0.11/0.26,Hey1-pasr 0.28/0, Hotair 0.25/0, Ins16 0.27/3.09, and Ccdc8 0.22/5.04.On this basis, we set the enrichment cutoff at 3:1. The combinedcriteria for transcript inclusion [RPKM(WT)≥0.4,RPKM(WT)/RPKM(null)≥3.0] are expected to eliminate false positives andsubtract background based on direct comparisons between WT and nulllibraries using an established set of controls. By these criteria, weestimate the PRC2 transcriptome at 9,788 RNAs (Table 2). Some 4,446transcripts in the joined UCSC transcriptome (39,003 transcripts) wereincluded in our PRC2 transcriptome (FIG. 2B). Another 3,106 UCSCtranscripts were hit but only on the reverse strand, implying theexistence of 3,106 previously unannotated antisense RNAs. Some 1,118UCSC transcripts were hit in both directions, implying the existence of2,236 additional distinct transcripts. 19% of reads did not have a hitin the UCSC database. These “orphan reads” suggest that thetranscriptome may include other novel transcripts. Therefore, 9,788represents a lower bound on the actual PRC2 transcriptome in ES cells.Because the total mouse transcriptome is believed to be anywhere from40,000 to 200,000, the PRC2 transcriptome comprises 5-25% of all mousetranscripts, depending on the actual size of the total transcriptome.

Example 3 Epigenetic Features

We examined specific epigenetic features (FIG. 2B, Tables I, 3 to 7).Interestingly, the RepA region within Xist and the 3′ end of Tsix wererepresented many times (FIG. 2C), a region consistent with the proposedEzh2 footprint (Zhao et al., 2008). In a metagene analysis, we queriedthe relationship of transcripts to transcription start sites (TSS) byplotting read numbers as a function of distance (FIG. 2D). On theforward strand, enrichment was observed at −2.0 to +0.001 kb; on thereverse strand, peaks were discernible at −0.5 to +0.1 kb. Theenrichment occurred above background (null, IgG controls) (FIG. 7C). TSSassociation is notable given the existence of short transcripts atpromoters (Kapranov et al., 2007; Core et al., 2008; Seila et al., 2008;Taft et al., 2009), PRC2's preferred occupancy near promoters (Boyer etal., 2006; Lee et al., 2006; Schwartz et al., 2006; Ku et al., 2008),and identification of several TSS-associated RNAs which bind PRC2(Kanhere et al., 2010).

We next asked how much of the PRC2 transcriptome intersects PRC2-bindingsites (Boyer et al., 2006; Lee et al., 2006) and bivalent domains in EScells (Bernstein et al., 2006a; Mikkelsen et al., 2007; Ku et al.,2008). Notably, 562 of 2,704 bivalent domains (21%) and 330 of 1,800Suz12-binding sites (18%) were hit by at least one RNA (FIG. 2B, Table3,4), raising the possibility that RNA may be involved in recruiting orretaining Polycomb complexes in a subset of binding sites and controlstem cell fate.

Sites which do not intersect our transcriptome may recruit PRC2 usingother mechanisms.

We also queried the extent of overlap with a group of intergenic ncRNAdubbed “lincRNA” (Guttman et al., 2009). Intersecting 2,127 mouselincRNA with our 9,788 transcripts revealed an overlap of 216 (FIG. 2B,Table 5), indicating that lincRNA account for ˜2% of the PRC2transcriptome. Of human lincRNA, 260 may have potential to associatewith PRC2 (Khalil et al., 2009). To ask whether the 260 human lincRNAoverlap with the 216 mouse lincRNA in our PRC2 transcriptome, we mappedsyntenic coordinates in the mouse by LiftOver (available on the worldwide web at genome.ucsc.edu/cgi-bin/hgLiftOver) but found norecognizable homology between the two subsets. Thus, our transcriptomerepresents a large and distinct set of PRC2-interacting RNAs.

Because misregulation of Polycomb proteins is often associated withcancer, we intersected PRC2-interacting RNAs with oncogene and tumorsuppressor loci (Sparmann and van Lohuizen, 2006; Bernardi and Pandolfi,2007; Miremadi et al., 2007; Rajasekhar and Begemann, 2007; Simon andLange, 2008). Intriguingly, of 441 oncogenes and 793 tumor suppressors(available on the world wide web at cbio.mskcc.org/CancerGenes), 182(41%) and 325 (41%) respectively have at least one PRC2-interactingtranscript of either orientation (FIG. 2B, Tables 6, 7), suggesting thatRNA may play a role in misregulating Polycomb recruitment in cancer.Notable examples include c-Myc, Brca1, Klf4, and Dnmt1.

Finally, like X-chromosome inactivation, genomic imprinting must beregulated in cis. Imprinted genes are controlled by a cis-acting‘imprinting control region’ (ICR) that dictates parent-specificexpression (Edwards and Ferguson-Smith, 2007; Thorvaldsen andBartolomei, 2007). Interestingly, ICRs are generally associated withlong transcripts (Williamson et al., 2006; Pandey et al., 2008; Wan andBartolomei, 2008), many of which were found in the PRC2 transcriptome(FIG. 2B, Table 2). They include H19, Gtl2, Kcnq1ot1, and Nespas.Multiple hits occurred in Nespas RNA/TR019501 (FIG. 3A), an antisenseRNA from the primary ICR thought to regulate the Nesp/Gnas cluster

(Coombes et al., 2003; Williamson et al., 2006). Also hit repeatedly wasGtl2 (FIG. 3B), the locus believed to control Dlk1 imprinting (Edwardset al., 2008), along with anti-Rtl1 and an antisense counterpart of Gtl2(here dubbed Gtl2-as). Hits within ICR-associated long transcripts hintthat RNA may regulate imprinted clusters by targeting PRC2.

Example 4 Validation of RNA-PRC2 Interactions

We next validated RNA-protein interactions by several approaches. First,we performed RIP-qPCR and found that candidate RNAs had significantenrichment in the α-Ezh2 relative to IgG pulldowns (FIG. 4A). Strongpositive pulldowns were observed for the imprinted Gtl2, its antisensepartner Gtl2-as/Rtl1, and Nespas/TR019501. A number of previouslyunknown antisense transcripts or RNAs linked to disease loci was alsoenriched, including Hspa1a-as (antisense to Hsp70), Malat-1-as(antisense to Malat-1), Bgn-as (antisense to Bgn), Ly6e-as (antisense tolymphocyte antigen 6 complex locus E), Foxn2-as (antisense to Foxn2),and an RNA upstream of Htr6 serotonin receptor. Second, we compared theamount of RNA pulled down by α-Ezh2 in WT versus Ezh2−/− ES cells (FIG.4B). In every case, the RNA was significantly more enriched in WT. Bycontrast, the negative control Malat-1 sense transcript showed noenrichment. Third, we performed UV-crosslink RIP, an alternative methodof testing RNA-protein interactions in vivo based on the ability of UVto covalently link RNA to protein at near-zero Angstroms (Ule et al.,2005). Because crosslinking occurs only at short range and complexes areisolated with disruptive sonication and high-salt washes, this methodbetter detects direct RNA-protein interactions and may avoidreassociation artifacts during RNA isolation. Enrichment of candidateRNAs was similarly observed using this method (FIG. 4C). Combined, thesedata support the specificity of RIP-seq and suggest direct interactionsbetween RNA and Ezh2.

Nearly half of the transcripts identified by RIP-seq were previouslyunannotated (FIG. 2B). To verify their existence, we performed Northernanalysis and found discrete transcripts in ES cells (FIG. 4D). Toconfirm the nature of nucleic acids precipitated by α-Ezh2, wepretreated nuclear extracts with RNases of different substratespecificities. Digesting with single-stranded RNase (RNase I) anddouble-stranded RNase (RNAse V1) abolished RNA pulldown, whereasdigesting with RNase H (which degrades the RNA strand in RNA:DNAhybrids) and DNase I had no effect (FIG. 4E). Thus, the RNAs in complexwith PRC2 have single- and double-stranded character.

Example 5 Direct Binding of RNA to PRC2

We next addressed whether RNA directly binds PRC2 by in vitrobiochemical analyses using purified recombinant human PRC2 subunits,EED, EZH2, SUZ12, and RBAP48 (FIG. 5A). The newly identified antisenseRNA for Hes1 (a transcription factor in the Notch signaling pathway(Axelson, 2004)) contains a double stem-loop structure, a motif alsofound in RepA (Zhao et al., 2008) (FIG. 5B). In an RNA electrophoreticmobility shift assay (EMSA), both the 28-nt RepA and 30-nt Hes1-asprobes were shifted by PRC2, whereas RNAs derived from other regions ofXist (DsI, DSII) were not. Mutating the stem-loop structures reducedPRC2 binding. To determine which subunit of PRC2 binds Hes1-as, weperformed EMSA using specific subunits (FIG. 5A, D, E). EZH2 stronglyshifted wildtype but not mutated Hes1-as RNA, whereas neither SUZ12 norEED shifted Hes1-as. The RNA-protein shift was always more discrete whenwhole PRC2 was used, suggesting that other subunits stabilize theinteraction. These results show that Hes1-as RNA directly andspecifically interacts with PRC2 and Ezh2 is the RNA-binding subunit. Wealso examined Gtl2 RNA. Because Gtl2 is 1.7-4.3 kb and too large to testby EMSA, we performed RNA pulldown assays (FIG. 5F). We invitro-transcribed Gtl2, a truncated form (1.0-kb from the 5′ end), RepA,and Xist exon 1 (negative control), and tested equal molar amounts ofeach RNA in pulldown assays using Flag-PRC2 or Flag-GFP proteins. Bothfull-length and truncated Gtl2 RNAs were consistently enriched in PRC2pulldowns. RepA RNA was also enriched, whereas Xist exon 1 was not.These results demonstrated that Gtl2 RNA—most likely its proximal 1.0kb—directly and specifically binds PRC2.

Example 6 Gtl2-PRC2 Interactions Regulate Gene Expression at Dlk1-Gtl2

To investigate whether RIP-seq succeeded in discovering new functions,we focused on Gtl2-PRC2 interactions at Dlk1-Gtl2, the imprinted diseaselocus linked to the sheep Callipyge (gluteal hypertrophy), murine growthdysregulation, and human cancers (Edwards et al., 2008; Takahashi etal., 2009). The maternally expressed Gtl2 is associated with the ICR(FIG. 6A) and has been proposed to regulate paternally expressed Dlk1(Lin et al., 2003; Takahashi et al., 2009), but the mechanism of actionis currently unknown. To determine if the Gtl2 transcript per seregulates Dlk1, we knocked down Gtl2 in ES cells and observed a 2-foldupregulation of Dlk1, consistent with the idea that Dlk1 changed frommono- to bi-allelic expression (FIG. 6B). Gtl2-as was also upregulated.Because shRNAs target RNA for degradation post-transcriptionally, theseexperiments demonstrate that Gtl2 functions as RNA.

To address if the RNA operates by attracting PRC2 to Dlk1, we carriedout quantitative chromatin immunoprecipitation (ChIP) using α-Ezh2 andα-H3-K27me3 antibodies. Indeed, when Gtl2 RNA was knocked down, wedetected a two-fold decrease in Ezh2 recruitment to the Dlk1 promoterand a commensurate decrease in H3-K27 trimethylation in cis (FIG. 6C),consistent with increased Dlk1 expression (FIG. 6B). We also sawdecreased Ezh2 recruitment and H3-K27 trimethylation at a differentiallymethylated region (DMR) of the ICR proximal to Gtl2, whereas lessereffects were seen at the distal DMR (FIG. 6C). Because the distal DMR isgenetically upstream of Gtl2 (Lin et al., 2003; Takahashi et al., 2009),we did not expect regulation by Gtl2. Gapdh and Actin controls did notshow significant decreases after Gtl2 knockdown, and decreased Ezh2recruitment to Dlk1 was not the result of generally decreased Ezh2levels in Gtl2-knockdown cells (FIG. 6D). These data argue that Gtl2indeed functions by attracting PRC2 to Dlk1. In further support,abolishing Ezh2 phenocopied the Gtl2 knockdown, with a ˜3-fold increasein Dlk1 expression relative to Gtl2 levels (FIG. 6E). Given directGtl2-PRC2 interactions (FIG. 5) and loss of Ezh2/H3-K27me3 at Dlk1 whenGtl2 is knocked down (FIG. 6), we conclude that Gtl2-PRC2 interactionsregulate Dlk1 by targeting PRC2 to Dlk1 in cis.

Example 7 Long ncRNA Modulation of Oncogenes and Tumor Suppressor Genes

As described hereinabove, application of the RIP-seq method generated agenome-wide pool of long ncRNA transcripts that bind to the PRC2Transcriptome. Genomic distribution of the identified transcripts wasexamined by plotting distinct reads as a function of chromosomeposition. As a result, lncRNAs that regulate both oncogenes and tumorsuppressor genes were identified. FIG. 13 depicts a plot showing theregion around the c-Myc oncogene (red bar). Zooming into the readsaround the c-Myc oncogene shows an impressive peak of PRC2 binding (tallred peak at chromosome coordinate 61,870,000). Further analysis revealedthis lncRNA to be Pvt1 (GenBank Accession Nos. Z12002.1 (mouse), or═NR_003367.1 (human)). Pvt1 is known in the art to be disrupted in somecases of Burkitt's lymphoma as well as in plasmacytomas (e.g., bytranslocations from another chromosome). Therefore, Pvt1 is likely toact by targeting PRC2 to c-Myc in order to repress its expression.Accordingly, exogenous administration of Pvt1 or fragments thereof couldrescue Pvt1 loss-of-function phenotypes contributing to various cancers.

FIG. 14 depicts a plot showing the region around the Nkx2-1 gene (alsoknown as Titf1; Genbank Acc. No. NM_001079668.2 (human mRNA) andNM_001146198.1 (mouse mRNA); genomic sequence is NC_000014.8 (human),NT_026437.12, NC_000078.5 (mouse) or NC_000078.5 (mouse)). In humans,NKX2-1 is frequently amplified or mutated in lung adenocarcinomas andhas been directly linked to lung oncogenesis. It is described as aproto-oncogene in driving initial cancer development, but at the sametime, its loss of expression is eventually associated with badprognosis. Therefore, regulation of NKX2-1 is of special interest, asits regulatory elements could be used to modulate NKX2-1 expression inpatients. The circled areas of the plot represent locations of PRC2binding to an antisense lncRNA within the mouse Nkx2-1 gene (a.k.a.Titf1). Based on the hit pattern and density, the antisense RNA resideswithin the interval on mouse Chr12 from bp 57,636,100 to 57,638,250(likely including the promoter of Nkx2-1 and AK14300) (SEQ ID NO:191088), in the mouse genome assembly version, NCBI37/mm9. The RNAspecies at the 5′ end of Nkx2-1 is a promoter-associated antisensetranscript overlapping the Nkx2-1 promoter and residing within abivalent domain. As noted above, mouse and human RNAs arewell-conserved, even for long ncRNAs (e.g., PVT1, XIST, GTL2).Mouse-to-human LiftOver analysis and analysis in the UCSC genome browserof syntenic positions indicate the existence of a similar noncoding,antisense promoter-associated transcript for the human NKX2-1/TITF1locus (likely overlapping, if not coincident, with Human gene BX161496;Chr14: bp 36,988,521-36,991,722 (SEQ ID NO: 191087) in human genomeassembly version, GRCh37/hg19). The similarity in gene structure and theexistence of an upstream RNA sequence are evident in the UCSC genomebrowser. These points suggest that regulation of the human locus may besimilar to that in mouse.

Levels of this antisense transcript can be modulated to affectexpression of NKX2-1. The promoter-associated antisense transcript areadministered to subjects, e.g., lung adenocarcinoma patients, withamplified NKX2-1 expression, and/or introduced into tumor cells, todecrease expression of NKX2-1. Alternatively, in patients with poorprognosis who have lost NKX2-1 expression, an inhibitory RNA, such as anLNA molecule that binds specifically to a region within the NKX2-1antisense lncRNA, is introduced to antagonize the PRC2-interactingantisense transcript and restart expression of the NKX2-1 gene.

Example 8 Identification of PRC2-binding Peaks from Appendix I

In some or any embodiments, the region of an RNA to which a proteinbinding partner (e.g., PRC2) binds is one of the exemplary locations ona target lncRNA to which an inhibitory nucleic acid is designed tohybridize. For example, these regions can be identified by reviewing thedata in Appendix I and identifying regions that are enriched in thedataset; these regions are likely to include PRC2-binding sequences.

The sequence reads in Appendix I come directly off the Illumina GA-IIgenome analyzer and are in an orientation that is the reverse complementof the PRC2-binding transcript. Appendix I is a filtered subset of allof the reads after bioinformatic filtering removed adaptor/primerdimers, mitochondrial RNA, rRNA, homopolymers, reads with indeterminatenucleotides, and truncated reads (<15 nt). They are likely to representregions best protected from endogenous nucleases during RIP andsubsequent RNA purification steps described in Example 1 above (aRIP-seq method) and thus represent candidate regions of RNA that bind toPRC2 or associated proteins or complexes. From Appendix I, reads wereextracted corresponding to transcripts that are enriched 3:1 in WT vs.null [RPKM(WT)/RPKM(null)≥3.0] and with a minimal RPKM value of 0.4. Wethen identified regions of the PRC2-binding transcripts with anuninterrupted pile-up of reads (peaks) and consider them candidate PRC2contact regions within the RNA

The sequence reads in Appendix I were used to generate sequence coverageon the reference genome using the Broad Institute's Arachne aligner,ShortQueryLookup, which is based on making a k-mer (K=12) dictionary ofthe reference genome and performing a local Smith-Waterman alignment ona read's candidate locations based on matching k-mer locations in thegenome. The aligner does multiple placements. The best alignment isallowed to have at most one error and alignments that differ from thebest alignment's number of errors by one are also accepted. The coverageis normalized by dividing by the number of places the read aligns (e.g.if a reads aligns to four places, 0.25 is added to each of the bases inthe four places).

To obtain the target Peaks, the following methodology was used. Abase-level mouse (mm9) coverage file of regions where the wild-typecoverage of the transcriptome is enriched at least three-fold over thecoverage of the Ezh2 −/− transcriptome and has a minimum RPKM coverageof at least 0.4 serves as the starting point. The coverage isstrand-specific. Next, in non-overlapping consecutive windows of 100 bpsin length, peak values and their locations are determined. Peakpositions are then corrected for those peaks that are on the edge of awindow that are determined to be on a side of a larger peak. Those peaksare moved to the top of the larger peak. Duplicate peak locations arethen removed. Peaks positions that are on a plateau are moved to thecenter of the plateau. The coverage is then smoothed using a Gaussiankernel, (1/sqrt(2*sigma*pi))*exp(−t2/(2*sigma)), where sigma=5.0. Peakwidths are then determined by locating the nearest position to the peaksuch that the smoothed coverage is less than or equal to one-third themaximum coverage. Adjacent peaks that overlap each other are resolved byplacing a boundary between them at the midpoint between the peaks.

Peaks are then output into a table with the position, width, the maximumamplitude, and the sum of unsmoothed coverage underneath the width ofthe peak. The corresponding nucleotide sequences of the mouse Peaks inmm9 (converted to RNA by replacing T with U) appear in the sequencelisting as SEQ ID NOS: 21583 to 124436, or 190717 to 190933.Mouse-to-human LiftOver of the mouse chromosome coordinates and strandof these mouse Peaks was performed in the UCSC genome browser asdescribed herein, to generate orthologous human chromosome coordinates.This process and LiftOver chains are generally described in Kent et al.,Proc. Nat'l Acad. Sci., 100(20) 11484-11489 (2003). When the mousecoordinates (mm9) of each mouse Peak were converted to the correspondinghuman (hg19) coordinates, mapping percentages of 50, 65, 75, and 95yielded essentially identical location and length results whenever amatch occurred. Consequently, the 50% mapping parameter was used.

Each corresponding human Peak RNA sequence (i.e., the nucleotidesequence of the human chromosomal coordinates and strand, converted toRNA by replacing T with U) appear in the sequence listing as SEQ ID NOS:124437 to 190716, or 190934 to 191086. These human Peaks and the humanPRC2 transcriptome (i.e. human sequences of PRC2-binding transcriptsreferenced in Tables 1-7) were intersected with known genes from theNCBI database to identify genes targeted by the PRC2-binding RNA (i.e.an intersecting or nearby gene).

Table 8 shows the annotation of the mouse and human Peaks with the namesof genes that were near or intersected with each Peak. The unique NCBIgene ID associated with the human gene (listed first) or mouse gene(listed second) appears in parentheses adjacent to the gene name. Thedegree of overlap between the Peak coordinates and the gene coordinatesappears in square brackets. A positive number indicates the number ofoverlapping nucleotides between the two, and a negative numberrepresents the size of the gap between the two (i.e. the number ofnucleotides of distance between the two). For Peaks, an “F” within thesquare brackets indicates that the Peak coordinates fully overlap thegene coordinates. For transcripts, an “F” within the square bracketsindicates that the transcript coordinates fully overlap the genecoordinates, or vice versa. The RNA transcript or Peak is “antisense” tothe reference genes in the “Opposite Strand” column, while the RNAtranscript or Peak is in the same “sense” orientation as the referencegene in the “Same Strand” column.

Bioinformatic analysis indicates that the average Peak is about 40-60bases, which is an excellent size for initial design of inhibitorynucleic acids. More than 100,000 Peaks were identified in the mousetranscriptome of Table 2. Each of these Peaks is fully represented bythe reverse-complement reads in Appendix I since it corresponds to asegment of overlapping reverse-complement reads from Appendix I. ThePeaks can be found anywhere within the coding gene, and in either senseor antisense orientations. Peaks can also be found in the promoter/5′UTRregions, introns, internal exons, and 3′UTR and beyond. The analysisstrongly suggests that the PRC2-interacting transcripts are not theprotein-coding mRNA, but a distinct transcript or transcripts thatoverlap with the mRNA sequence. Many are novel RNAs not previouslydescribed.

Routine methods can be used to design an inhibitory nucleic acid thatbinds to target locations or segments with sufficient specificity, orare sufficiently complementary to the target RNA to give the desiredeffect. In some embodiments, the methods include using bioinformaticsmethods known in the art to identify regions of secondary structure,e.g., one, two, or more stem-loop structures, or pseudoknots, andselecting those regions to target with an inhibitory nucleic acid.

Additional target segments 5-500 nucleotides in length, or about 5 toabout 100 nucleotides in length, comprising a stretch of at least five(5) consecutive nucleotides within the Peak, or immediately adjacentthereto, are considered to be suitable for targeting as well.

Example 9 In Vitro Effect of Inhibitory Oligonucleotides on Upregulationof mRNA Expression

A. ApoE

Inhibitory oligonucleotides were designed to target lncRNA as set forthin Table 8 in order to upregulate ApoE. The oligonucleotides were lessthan 16 bases in length and comprised unmodified DNA and multiple lockednucleic acid modified bases, all linked by phosphorothioate bonds.Transfection and data analysis were carried out briefly as follows.

RNA was harvested from the Hep 3B cells using Promega SV 96 Total RNAIsolation system omitting the DNAse step. In separate pilot experiments,50 ng of RNA was determined to be sufficient template for the reversetranscriptase reaction. RNA harvested from the Hep3B cells wasnormalized so that 50 ng of RNA was input to each reverse transcriptionreaction. For the few samples that were too dilute to reach this limit,the maximum input volume was added. Quantitative PCR evaluation was thencompleted.

A baseline level of ApoE mRNA expression was determined throughquantitative PCR as outlined above. Baseline levels were also determinedfor mRNA of various housekeeping genes which are constitutivelyexpressed. A “control” housekeeping gene with approximately the samelevel of baseline expression as ApoE mRNA was chosen for comparisonpurposes to ApoE.

Hep3B cells were seeded into each well of 24-well plates at a density of25,000 cells per 500 uL and transfections were performed withLipofectamine and the inhibitory oligonucleotides. Control wellscontained Lipofectamine alone. At 48 hours post-transfection,approximately 200 uL of cell culture supernatants were stored at −80 Cfor ELISA. At 48 hours post-transfection, RNA was harvested from the Hep3B cells and quantitative PCR was carried out as outlined above. Thepercent induction of ApoE mRNA expression by each inhibitoryoligonucleotide was determined by normalizing mRNA levels in thepresence of the inhibitory oligonucleotide to the mRNA levels in thepresence of control (Lipofectamine alone). This was comparedside-by-side with the increase in mRNA expression of the “control”housekeeping gene.

A total of 26 oligonucleotides tested were complementary to SEQ ID NO:15050 in Table 2. Of these 26 oligonucleotides, 7 upregulated apoEexpression in human Hep3B cells, as indicated by increased ApoE mRNAlevels relative to the “control” housekeeping gene.

The above procedure was repeated using human renal proximal tubuleepithelial cells (RPTEC). Of the 26 oligonucleotides complementary toSEQ ID NO: 15050 in Table 2, 5 increased ApoE mRNA levels in renalcells, relative to the “control” housekeeping gene. Levels increased byabout 1.5 to about 5-fold over baseline expression.

In addition, of 11 oligonucleotides that are complementary to Peaks inTable 8 associated with apoE, 3 upregulated apoE expression.

Inhibitory oligonucleotides as short as 8 nucleobases in length weredemonstrated to upregulate gene expression.

B. Nkx2-1

The experiments as described in Example 9A above were repeated forinhibitory oligonucleotides designed to target lncRNA as set forth inTable 8 in order to upregulate Nkx2-1. A total of 13 oligonucleotidestested were complementary to SEQ ID NO. 17040 in Table 2. Of these 13oligonucleotides, 3 upregulated Nkx2-1 expression as indicated byincreased Nkx2-1 mRNA expression relative to baseline, although no“control” housekeeping gene could be matched with Nkx2-1 due to lowlevels of intrinsic expression. In addition, of 9 oligonucleotides thatare complementary to Peaks in Table 8 associated with Nkx2-1, 3upregulated Nkx-21 expression.

C. Brca1

The experiments as described in Example 9A above were repeated forinhibitory oligonucleotides designed to target lncRNA as set forth inTable 8 in order to upregulate Brca1. A total of 30 oligonucleotidestested were complementary to SEQ ID NOs: 192,309 in Table 2 and SEQ IDNO: 192,965. Of these 30 oligonucleotides, 5 oligonucleotidesupregulated Brca1 expression. Of these 30 oligonucleotides, 13oligonucleotides were also complementary to Peaks in Table 8 associatedwith Brca1. Of these 13 oligonucleotides complementary to Peaks, 2oligonucleotides upregulated Brca1 expression. Levels increased by about2 to about 3 fold over baseline expression.

D. Smad7

The experiments as described in Example 9A above were repeated forinhibitory oligonucleotides designed to target lncRNA as set forth inTable 8 in order to upregulate Smad7, with the following exception: thekidney cell line RPTEC was used instead of HepB3. A total of 28oligonucleotides tested were complementary to SEQ ID NO. 18602 in Table2. Of these 28 oligonucleotides, 4 upregulated Smad7 expression. Inaddition, of 28 oligonucleotides that are complementary to Peaks inTable 8 associated with Smad7, 4 upregulated Smad7 expression.

E. SirT6

The experiments as described in Example 9A above were repeated forinhibitory oligonucleotides designed to target lncRNA as set forth inTable 8 in order to upregulate SirT6. A total of 25 oligonucleotidestested were complementary to SEQ ID NO: 192,182 in Table 2. Of these 25oligonucleotides, 3 upregulated SirT6 expression. A total of 2oligonucleotides tested were complementary to SEQ ID NO: 130,694 inTable 2. Of these 2 oligonucleotides, 1 upregulated SirT6 expression. Atotal of 2 oligonucleotides tested were complementary to SEQ ID NO:130,695 in Table 2. Of these 2 oligonucleotides, neither upregulatedSirT6 expression. Levels increased by 2 to 6 fold over baselineexpression. In addition, of 6 oligonucleotides that are complementary toPeaks in Table 8 associated with SirT6, 1 upregulated SirT6 expression.

F. Serpinf1

The experiments as described in Example 9A above were repeated forinhibitory oligonucleotides designed to target lncRNA as set forth inTable 8 in order to upregulate Serpinf1. A total of 38 oligonucleotidestested were complementary to SEQ ID NOs: 16698 and 16699 in Table 2. Ofthese 38 oligonucleotides, 3 upregulated SerpinF1 expression. Levelsincreased by 1.2 to 2 fold over baseline expression. In addition, of 32oligonucleotides that are complementary to Peaks in Table 8 associatedwith Serpinf1, 3 upregulated SerpinF1 expression.

Example 10 LNA Molecules Targeting Xist Repeat C rapidly displace XistRNA from Xi

Repeat C was aligned using Geneious (Drummond et al., (2010) Geneiousv5.1, Available on the internet at geneious.com) and LNA moleculescomplementary to two regions with a high degree of inter-repeatconservation were synthesized (FIG. 15A). The first LNA molecule showedconservation in all 14 repeats (LNA-C1) and the second in 13 of 14(LNA-C2) (FIG. 15A). LNA molecules were nucleofected separately intotransformed mouse embryonic fibroblasts (MEFs), and the cells wereadhered onto slides and fixed in situ at various timepoints between 0minutes (immediately after nucleofection) and 8 hourspost-nucleofection. To examine effects on Xist RNA, RNA fluorescence insitu hybridization (FISH) was performed using Xist-specific probes. (MEFcells are tetraploid due to transformation; each tetraploid cell has twoXa and two Xi). In controls transfected with scrambled LNA molecules(LNA-Scr), robust Xist clouds were seen in 80-90% of cells at alltimepoints (FIG. 15C). Intriguingly, introduction of either LNA-C1 or-C2 resulted in immediate loss of Xist RNA from Xi (FIG. 15B; LNA-C1shown, with similar results for LNA-C1 and LNA-C2). Even at t=0 (cellsfixed immediately, within seconds to minutes, after LNA introduction),˜10% of nuclei displayed a loosening of the Xist RNA clusters, with theclusters appearing faint and diffuse (FIG. 15C lightest grey bars)(n=149). The percentage of nuclei with full Xist clouds continued todrop during the first hour and reached a minimum at t=60 minutes (21%,n=190). These findings indicate that LNA molecules disrupted Xistbinding to chromatin as soon as they were introduced. However, the lossof Xist from Xi was transient, as pinpoints of Xist RNA typical ofnascent transcripts seen in undifferentiated embryonic stem (ES) cells,became visible at t=3 hr (FIG. 15C, darkest grey bars) (18%, n=190 at 1hr; 36%, n=123 at 3 hr). Full recovery of Xist clouds was not seen until8-24 hr post-nucleofection (81% at 8 hr, n=117).

The next experiment addressed whether LNA molecules had similar effectsin mouse ES cells an established ex vivo model which recapitulates XCIas the cells differentiate in culture. In the undifferentiated state,wildtype female ES cells express low levels of Xist RNA, visible aspinpoint signals by RNA FISH. By day 6 of differentiation, ˜40% of cellswould normally have upregulated Xist RNA. When ES cells werenucleofected with LNA-C1 on day 6, Xist displacement occurred rapidly,reaching a maximum at 1 hr and recovering by 8 hr. Thus, LNA moleculeswere effective in ES cells as well as in somatic cells. These resultscontracted sharply with those obtained from MEFs nucleofected withsiRNAs or shRNAs toward the same region of Xist. Neither siRNAs norshRNAs led to loss of Xist at the 1,3 or 24 hour timepoints, and partialdecreases in Xist clouds occurred only at 48 hours (83%, n=84 at 1 hr;80%, n=106 at 24 hr). Thus, LNA molecules can be used efficiently totarget long nuclear ncRNAs such as Xist with extremely rapid kinetics,much more rapid than the action of siRNAs or shRNAs, in multiple celltypes.

To test the specificity of the LNA molecules, human 293 cells werenucleofected with the Repeat C LNA molecules. Sequence comparisonbetween the mouse and human Xist/XIST revealed that the region targetedby LNA-C1 is conserved in 10 of 15 nt and is conserved in 10 of 14 ntfor LNA-C2 (FIG. 15C). Nucleofection of scrambled LNA molecules followedby XIST RNA FISH in human cells showed two normal XIST clouds in nearlyall cells (92%, n=108). Similarly, nucleofection with either LNA-C1 orLNAC-2 did not change the XIST clouds (LNA-C1, 89%, n=126; LNA-C2, 85%,n=139). Thus, mouse Repeat C LNA molecules do not affect human XISTlocalization, suggesting that they function in a species-specificmanner. To determine whether human Repeat C could displace human XIST,we nucleofected LNA molecules complementary to the human Repeat C into293 cells, but observed no loss of XIST clouds (91%, n=103 at 1 hr; 87%,n=95 at 3 hr and 92%, n=85 at 8 hr). This finding indicated that,although Repeat C may play a role in humans, additional human elementsfunction in RNA localization. Whereas mouse Repeat C occurs 14 times,the human repeat is present only once (8, 9).

Example 11 Xist RNA is Displaced without Transcript Destabilization

Several mechanisms could explain the disappearance of Xist. LNAmolecules could anneal to the complementary region and target Xist fordegradation. Alternatively, hybridization to LNA molecules coulddisplace Xist RNA from Xi without affecting the transcript stability. Todistinguish between these possibilities, Xist levels were quantitatedrelative to Gadph levels (control) by qRT-PCR at different timepoints.At 1 hr when Xist clouds were no longer visible, Xist levels remainedcomparable to that seen in the scrambled control (FIG. 16). Even at 3and 8 hr, Xist levels did not change significantly. These results showedthat displacement of Xist occurred without complete RNA degradation.Thus, LNA molecules function by blocking Xist interaction with chromatinrather than altering the RNA's stability.

The rapid displacement of Xist and the slow kinetics of recoveryprovided the opportunity to investigate several unanswered questionsregarding Xist's mechanism of localization. To ask whether reappearanceof Xist on Xi is due to relocalization of displaced Xist molecules or tocoating by newly synthesized RNA, we performed time-course analysis inthe presence of actinomycin D (ActD), an inhibitor of RNA polymerase II.Previous studies have shown that the half-life of Xist in the cell isapproximately 4-6 hr (14-16). It was reasoned that treating cells withActD for 0-8 hr would prevent new synthesis of Xist RNA during thistimeframe and that, therefore, reappearance of Xist clouds would implyrelocalization of displaced RNA back onto Xi. LNA molecules wereintroduced into cells and then the cells were allowed to recover inmedium containing ActD. In the scrambled controls, Xist clouds wereclearly visible at all time points without ActD. With ActD, Xist cloudswere apparent in the 1 and 3 hr timepoints and were lost by 8 hr,consistent with a 4-6 hr half-life. In LNA-C1- or LNA-C2-treated samplesallowed to recover without ActD, pinpoints of Xist were visible at 3 hrand Xist clouds were restored by the 8 hr timepoint. However, with ActD,Xist clouds were never restored, neither fully nor partially. Thus, Xistrecovery after LNA molecule-mediated displacement from Xi is due to newRNA synthesis and not relocalization of the displaced transcript.

Example 12 Xist RNA Localizes Near the X-Inactivation Center First

Taking further advantage of the rapid displacement and slow recovery,the long-standing question of whether Xist spreads in a piecemealfashion or localizes simultaneously throughout Xi was asked. Onehypothesis is that coating initiates near the Xist locus and proceeds toboth ends of the chromosome through booster elements located along the X(17). Alternatively, coating can occur all at once through multipleX-linked seeding points which would promote local spreading. Xistlocalization on metaphase chromosomes was analyzed during the 3-8 hrperiod of recovery. In cells treated with scrambled LNA molecules, allmetaphase chromosomes coated with Xist RNA showed a banded patternsimilar to the heterogeneous patterns described in earlier works(18-20). By contrast, LNA-C1 treated cells gave intermediate patterns.At 1 hr, no metaphase chromosomes showed a coat of Xist RNA (0%, n=41).At 3 hr when Xist RNA could be seen as a pinpoint in interphase cells,the predominant pattern was a combination of a single bright band in themiddle of the metaphase chromosome together with a small number of veryfaint bands elsewhere on the X (52%, n=46). This result suggested thatXist RNA initially bound locally. To determine whether the strong RNAband was localized to the Xist region, Xist RNA FISH was carried out onnon-denatured nuclei and followed with denaturation and hybridization toan Xist probe. Indeed, the focal RNA band observed at the 3-hr markcolocalized with the Xist region. At 5 hr, intermediate degrees ofcoating and intensities could be seen (68%, n=38). At 8 hr, thepredominant pattern was the whole-chromosome painting pattern typical ofcontrol cells (78%, n=38). In controls, intermediate patterns were notobserved at any time. These findings argue that Xist RNA initially bindsnearby, but seems to spread to the rest of Xi at the same time, withinthe temporal and spatial resolution of the FISH technique.

Example 13 Xist RNA Displacement is Accompanied by Loss of PRC2Localization

The pattern of Polycomb repressive complex 2 (PRC2) binding to Xi hasbeen of considerable interest, as its Ezh2 subunit catalyzestrimethylation of Histone H3 at lysine 27 (H3K27me3). Several studieshave shown that PRC2 localizes to Xi in an Xist-dependent manner, asdeleting Xist in ES cells precludes PRC2 recruitment duringdifferentiation and conditionally deleting Xist in MEF cells results inloss of PRC2 on Xi (21-24). However, the kinetics with which PRC2 isrecruited to and lost from X are not known. Because Xist RNA directlyrecruits PRC2 (12), it was asked whether LNA molecule-mediateddisplacement of Xist results in immediate loss of PRC2 by immunostainingfor Ezh2 in MEFs after LNA molecule delivery. Upon treatment with theRepeat C LNA molecules, Ezh2 was rapidly lost. There was nearly perfectconcordance between Xist and PRC2 loss. At 1 and 3 hr, Ezh2 foci werenever observed in nuclei that had lost Xist and, conversely, were alwaysobserved in nuclei with restored Xist clouds. The loss of Ezh2 on Xi wasdue to Ezh2 protein turnover (see Western analysis below). Transientdisplacement of PRC2, however, does not lead to appreciable H3K27me3loss within the 1-8 hr timeframe. Thus, PRC2's localization onto Xiabsolutely depends on Xist RNA for both initial targeting and for stableassociation after XCI is established, but the H3K27me3 mark is stable inthe short term when Xist and PRC2 are displaced.

Given this, it was asked whether LNA molecules affected gene silencing.At 3 hr when Xist was maximally displaced, RNA FISH was performed forXist and either Pgk1 or Hprt, two X-linked genes subject to XCI. Incontrol-nucleofected (LNA-Scr) cells, Xist clouds were observed from Xiand nascent Pgkl or Hprt transcripts from Xa. Nucleofection with LNA-1and LNA-4978 did not change the expression pattern, as two foci of Pgk1transcripts were still seen in 79% (n=39) of controls and 80% (n=36) ofLNA-C 1-treated cells, and two foci of Hprt RNA were seen in 84% (n=44)of controls and 79% (n=35) of LNA-C1-treated cells. Four foci of Pgk1 orHprt transcripts were never seen. Thus, consistent with retention ofH3K27me3, silencing was not disrupted by transient loss of Xist andPRC2.

Example 14 A Broader Domain around Repeat C is Required for XistLocalization

4The next experiments investigated other conserved repeats within Xist.As Repeat A has already been shown to be essential for targeting PRC2,the experiments focused on Repeats B, E, and F, and found that Xistlocalization was not affected by targeting any repeat individually or incombination (FIG. 17A). Conserved unique regions of Xist were alsotested, including LNA-726 (between Repeats A and F), LNA-4978 andLNA-5205 (between Repeats C and D), and LNA-3′ (distal terminus of Xist)(FIG. 17A). None affected Xist localization except for LNA-4978, whichcorresponds to a 15-nt element located 280 bp downstream of Repeat C.LNA-4978 induced effects similar to LNA-C1/C2 but differed by its slowerkinetics. At 1 hr, Xist clouds were still visible but appeared faint anddispersed (78%, n=125). The number of clouds reached a minimum at 3 hr(25%, n=158). At 8 hr, Xist was visible as small pinpoints (39%, n=123).Recovery was not complete until the 24-hr timepoint. As for Repeat C LNAmolecules, loss of Xist was not due to RNA turnover, as determined byqRT-PCR (FIG. 17B), and Ezh2 was displaced without affecting H3K27me3 orchange in Ezh2 protein level (FIG. 17C). Therefore, Xist localization tochromatin involves a broader region encompass both Repeat C and a uniqueregion directly downstream of the repeat.

To determine if the two motifs cooperate, LNA-4978 and LNA-C1 werenucleofected separately or together into MEFs. As expected, treatingwith LNA-C1 alone resulted in loss of Xist RNA clouds by 1 hr andrecovery beginning at 3 hr, and treating with LNA-4978 showed loss andrecovery at 3 hr and 8 hr, respectively. Treating with both LNAmolecules expanded the window of Xist depletion: Loss of Xist RNA andEzh2 was observed by 1 hr (as was the case for LNA-C1 alone) andrecovery did not begin until the 8 hr timepoint (as was the case forLNA-4978 alone). Thus, the LNA molecule effects were additive, notsynergistic, as the effects were not enhanced beyond the widening of theXist-depleted time window.

Example 15 Ezh2 Recovery after LNA Molecule Nucleofection is Slow butUniform along Xi

Finally, it was asked whether Ezh2 retargeting to Xi closely follows thepiecemeal relocalization of Xist RNA during the recovery phase. BecausePRC2 generally binds near promoters (25, 26), Ezh2 localization atX-gene promoters was analyzed by quantitative chromatinimmunoprecipitation (qChIP) (FIG. 18A). Although female cells have twoXs and Ezh2 epitopes pulled down by the antibody could theoreticallycome from either Xa or Xi, evidence indicates that the vast bulk of Ezh2and H3K27me3 is bound to Xi (21-24). Ezh2 was indeed enriched atpromoters of genes that are silenced on Xi (e.g., Xmr, Pgk1), but not atpromoters of genes (e.g., Jarid1c) that escape XCI (FIG. 18B). Then, MEFcells were nucleofected with LNA-C1 and performed qChIP using anti-Ezh2antibodies between 1 and 24 hr. At t=1 hr, Ezh2 levels decreaseddramatically at all tested target gene promoters to background levels(FIG. 18C), indicating that depletion of promoter-bound Ezh2 closelyfollowed Xist displacement along Xi. At the 3- and 8-hr points, therewas a gradual, uniform increase in Ezh2 levels across all genes, withmany genes appearing to have reached saturating amounts of Ezh2 byt=8hr. On promoters with the highest levels of Ezh2 at t=0 hr (FIG.18B), Ezh2 levels did not fully recover until 24 hr (FIG. 18C). Thus,ChIP pulldowns were expected to originate predominantly, if not nearlyexclusively, from Xi. In contrast, Ezh2 levels at the En1 control, aknown autosomal PRC2 target (27), did not change significantly (FIG.18D). Thus, Ezh2 levels fall and rise with similar kinetics throughoutXi. The loss of Xist RNA and Ezh2 binding between 1 and 8 hrs presents awindow of opportunity during which cells could be reprogrammed toachieve novel epigenetic states.

References 1. Kapranov P, Willingham A T, & Gingeras T R (2007)Genome-wide transcription and the implications for genomic organization.Nat Rev Genet 8(6):413-423.

2. Mercer T R, Dinger M E, & Mattick J S (2009) Long non-coding RNAs:insights into functions. Nat Rev Genet 10(3):155-159.

3. Krutzfeldt J, et al. (2005) Silencing of microRNAs in vivo with‘antagomirs’. Nature 438(7068):685-689.

4. Orom UA, Kauppinen S, & Lund A H (2006) LNA-modified oligonucleotidesmediate specific inhibition of microRNA function. Gene 372:137-141.

5. Morris K V (2008) RNA-mediated transcriptional gene silencing inhuman cells. Curr Top Microbiol Immunol 320:211-224.

6. Petersen M & Wengel J (2003) LNA: a versatile tool for therapeuticsand genomics. Trends Biotechnol 21(2):74-81.

7. Penny G D, Kay G F, Sheardown S A, Rastan S, & Brockdorff N (1996)Requirement for Xist in X chromosome inactivation. Nature379(6561):131-137.

8. Brockdorff N, et al. (1992) The product of the mouse Xist gene is a15 kb inactive X-specific transcript containing no conserved ORF andlocated in the nucleus. Cell 71(3):515-526.

9. Brown C J, et al. (1992) The human XIST gene: analysis of a 17 kbinactive X-specific RNA that contains conserved repeats and is highlylocalized within the nucleus. Cell 71(3):527-542.

10. Clemson C M, McNeil J A, Willard H F, & Lawrence J B (1996) XIST RNApaints the inactive X chromosome at interphase: evidence for a novel RNAinvolved in nuclear/chromosome structure. J Cell Biol 132(3):259-275.

11. Wutz A, Rasmussen T P, & Jaenisch R (2002) Chromosomal silencing andlocalization are mediated by different domains of Xist RNA. Nat Genet30(2):167-174.

12. Zhao J, Sun B K, Erwin J A, Song J J, & Lee J T (2008) Polycombproteins targeted by a short repeat RNA to the mouse X chromosome.Science 322(5902):750-756.

13. Beletskii A, Hong Y K, Pehrson J, Egholm M, & Strauss W M (2001) PNAinterference mapping demonstrates functional domains in the noncodingRNA Xist. Proc Natl Acad Sci USA 98(16):9215-9220.

14. Sheardown S A, et al. (1997) Stabilization of Xist RNA mediatesinitiation of X chromosome inactivation. Cell 91(1):99-107.

15. Sun B K, Deaton A M, & Lee J T (2006) A transient heterochromaticstate in Xist preempts X inactivation choice without RNA stabilization.Mol Cell 21(5):617-628.

16. Panning B, Dausman J, & Jaenisch R (1997) X chromosome inactivationis mediated by Xist RNA stabilization. Cell 90(5):907-916.

17. Gartler S M & Riggs A D (1983) Mammalian X-chromosome inactivation.Annu Rev Genet 17:155-190.

18. Duthie S M, et al. (1999) Xist RNA exhibits a banded localization onthe inactive X chromosome and is excluded from autosomal material incis. Hum Mol Genet 8(2):195-204.

19. Chadwick B P & Willard H F (2004) Multiple spatially distinct typesof facultative heterochromatin on the human inactive X chromosome. ProcNatl Acad Sci U S A 101(50):17450-17455.

20. Clemson C M, Hall LL, Byron M, McNeil J, & Lawrence J B (2006) The Xchromosome is organized into a gene-rich outer rim and an internal corecontaining silenced nongenic sequences. Proc Natl Acad Sci USA103(20):7688-7693.

21. Plath K, et al. (2003) Role of histone H3 lysine 27 methylation in Xinactivation. Science 300(5616): 131-135.

22. Kohlmaier A, et al. (2004) A chromosomal memory triggered by Xistregulates histone methylation in X inactivation. PLoS Biol 2(7):E171.

23. Silva J, et al. (2003) Establishment of histone h3 methylation onthe inactive X chromosome requires transient recruitment of Eed-Enx1polycomb group complexes. Dev Cell 4(4):481-495.

24. Zhang L F, Huynh K D, & Lee J T (2007) Perinucleolar targeting ofthe inactive X during S phase: evidence for a role in the maintenance ofsilencing. Cell 129(4):693-706.

25. Boyer LA, et al. (2006) Polycomb complexes repress developmentalregulators in murine embryonic stem cells. Nature 441(7091):349-353.

26. Ku M, et al. (2008) Genomewide analysis of PRC1 and PRC2 occupancyidentifies two classes of bivalent domains. PLoS Genet 4(10):e1000242.

27. Bracken A P, Dietrich N, Pasini D, Hansen K H, & Helin K (2006)Genome-wide mapping of Polycomb target genes unravels their roles incell fate transitions. Genes Dev 20(9):1123-1136.

28. Blais A, et al. (2005) An initial blueprint for myogenicdifferentiation. Genes Dev 19(5):553-569.

Axelson, H. (2004). The Notch signaling cascade in neuroblastoma: roleof the basic helix-loop-helix proteins HASH-1 and HES-1. Cancer Lett204, 171-178

Batzoglou, S., Jaffe, D. B., Stanley, K., Butler, J., Gnerre, S.,Mauceli, E., Berger, B., Mesirov, J. P., and Lander, E. S. (2002).ARACHNE: a whole-genome shotgun assembler. Genome Res 12, 177-189

Bernardi, R., and Pandolfi, P. P. (2007). Structure, dynamics andfunctions of promyelocytic leukaemia nuclear bodies. Nat Rev Mol CellBiol 8, 1006-1016

Bernstein, B. E., Mikkelsen, T. S., Xie, X., Kamal, M., Huebert, D. J.,Cuff, J., Fry, B., Meissner, A., Wernig, M., Plath, K., et al. (2006a).A bivalent chromatin structure marks key developmental genes inembryonic stem cells. Cell 125, 315-326

Bernstein, E., and Allis, C. D. (2005). RNA meets chromatin. Genes Dev19, 1635-1655

Bernstein, E., Duncan, E. M., Masui, O., Gil, J., Heard, E., and Allis,C. D. (2006b). Mouse polycomb proteins bind differentially to methylatedhistone H3 and RNA and are enriched in facultative heterochromatin. MolCell Biol 26, 2560-2569

Boyer, L. A., Plath, K., Zeitlinger, J., Brambrink, T., Medeiros, L. A.,Lee, T. I., Levine, S. S., Wernig, M., Tajonar, A., Ray, M. K., et al.(2006). Polycomb complexes repress developmental regulators in murineembryonic stem cells. Nature 441, 349-353

Carninci, P., Kasukawa, T., Katayama, S., Gough, J., Frith, M. C.,Maeda, N., Oyama, R., Ravasi, T., Lenhard, B., Wells, C., et al. (2005).The transcriptional landscape of the mammalian genome. Science 309,1559-1563

Cloonan, N., Forrest, A. R., Kolle, G, Gardiner, B. B., Faulkner, G J.,Brown, M. K., Taylor, D. F., Steptoe, A. L., Wani, S., Bethel, G, et al.(2008). Stem cell transcriptome profiling via massive-scale mRNAsequencing. Nat Methods 5, 613-619

Coombes, C., Arnaud, P., Gordon, E., Dean, W., Coar, E. A., Williamson,C. M., Feil, R., Peters, J., and Kelsey, G (2003). Epigenetic propertiesand identification of an imprint mark in the Nesp-Gnasxl domain of themouse Gnas imprinted locus. Mol Cell Biol 23, 5475-5488

Core, L. J., Waterfall, J. J., and Lis, J. T. (2008). Nascent RNAsequencing reveals widespread pausing and divergent initiation at humanpromoters. Science 322, 1845-1848

Denisenko, O., Shnyreva, M., Suzuki, H., and Bomsztyk, K. (1998). Pointmutations in the WD40 domain of Eed block its interaction with Ezh2. MolCell Biol 18, 5634-5642

Edwards, C. A., and Ferguson-Smith, A. C. (2007). Mechanisms regulatingimprinted genes in clusters. Curr Opin Cell Biol 19, 281-289

Edwards, C. A., Mungall, A. J., Matthews, L., Ryder, E., Gray, D. J.,Pask, A. J., Shaw, G, Graves, J. A., Rogers, J., Dunham, I., et al.(2008). The evolution of the DLK1-DIO3 imprinted domain in mammals. PLoSBiol 6, e135

Francis, N. J., Saurin, A. J., Shao, Z., and Kingston, R. E. (2001).Reconstitution of a functional core polycomb repressive complex. MolCell 8, 545-556

Gupta, R. A., Shah, N., Wang, K. C., Kim, J., Horlings, H. M., Wong, D.J., Tsai, M. C., Hung, T., Argani, P., Rinn, J. L., et al. (2010). Longnon-coding RNA HOTAIR reprograms chromatin state to promote cancermetastasis. Nature 464, 1071-1076

Guttman, M., Amit, I., Garber, M., French, C., Lin, M. F., Feldser, D.,Huarte, M., Zuk, O., Carey, B. W., Cassady, J. P., et al. (2009).Chromatin signature reveals over a thousand highly conserved largenon-coding RNAs in mammals. Nature

Kanhere, A., Viiri, K., Araujo, C. C., Rasaiyaah, J., Bouwman, R. D.,Whyte, W. A., Pereira, C. F., Brookes, E., Walker, K., Bell, G W., etal. (2010). Short RNAs Are Transcribed from Repressed Polycomb TargetGenes and Interact with Polycomb Repressive Complex-2. Mol Cell 38,675-688

Kapranov, P., Cheng, J., Dike, S., Nix, D. A., Duttagupta, R.,Willingham, A. T., Stadler, P. F., Hertel, J., Hackermuller, J.,Hofacker, I. L., et al. (2007). RNA maps reveal new RNA classes and apossible function for pervasive transcription. Science 316, 1484-1488

Khalil, A. M., Guttman, M., Huarte, M., Garber, M., Raj, A., RiveaMorales, D., Thomas, K., Presser, A., Bernstein, B. E., van Oudenaarden,A., et al. (2009). Many human large intergenic noncoding RNAs associatewith chromatin-modifying complexes and affect gene expression. Proc NatlAcad Sci U S A

Ku, M., Koche, R .P., Rheinbay, E., Mendenhall, E. M., Endoh, M.,Mikkelsen, T. S., Presser, A., Nusbaum, C., Xie, X., Chi, A.S., et al.(2008). Genomewide analysis of PRC1 and PRC2 occupancy identifies twoclasses of bivalent domains. PLoS Genet 4, e1000242

Lee, J. T. (2009). Lessons from X-chromosome inactivation: long ncRNA asguides and tethers to the epigenome. Genes Dev 23, 1831-1842

Lee, J. T. (2010). The X as model for RNA's niche in epigenomicregulation. Cold Spring Harb Perspect Biol 2, a003749

Lee, J. T., and Lu, N. (1999). Targeted mutagenesis of Tsix leads tononrandom X inactivation. Cell 99, 47-57

Lee, T. I., Jenner, R. G, Boyer, L. A., Guenther, M. G, Levine, S. S.,Kumar, R. M.,

Chevalier, B., Johnstone, S. E., Cole, M. F., Isono, K., et al. (2006).Control of developmental regulators by Polycomb in human embryonic stemcells. Cell 125, 301-313

Li, G, Margueron, R., Ku, M., Chambon, P., Bernstein, B. E., andReinberg, D. Jarid2 and PRC2, partners in regulating gene expression.Genes Dev 24, 368-380

Li, G, Margueron, R., Ku, M., Chambon, P., Bernstein, B. E., andReinberg, D. (2010). Jarid2 and PRC2, partners in regulating geneexpression. Genes Dev 24, 368-380

Lin, S. P., Youngson, N., Takada, S., Seitz, H., Reik, W., Paulsen, M.,Cavaille, J., and Ferguson-Smith, A. C. (2003). Asymmetric regulation ofimprinting on the maternal and paternal chromosomes at the Dlk1-Gtl2imprinted cluster on mouse chromosome 12. Nat Genet 35, 97-102

Mercer, T. R., Dinger, M. E., and Mattick, J. S. (2009). Long non-codingRNAs: insights into functions. Nat Rev Genet 10, 155-159

Mikkelsen, T. S., Ku, M., Jaffe, D. B., Issac, B., Lieberman, E.,Giannoukos, G, Alvarez, P., Brockman, W., Kim, T. K., Koche, R. P., etal. (2007). Genome-wide maps of chromatin state in pluripotent andlineage-committed cells. Nature 448, 553-560

Miremadi, A., Oestergaard, M. Z., Pharoah, P. D., and Caldas, C. (2007).Cancer genetics of epigenetic genes. Hum Mol Genet 16 Spec No 1, R28-49

Montgomery, N. D., Yee, D., Chen, A., Kalantry, S., Chamberlain, S. J.,Otte, A. P., and Magnuson, T. (2005). The murine polycomb group proteinEed is required for global histone H3 lysine-27 methylation. Curr Biol15, 942-947

Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., and Wold, B.(2008). Mapping and quantifying mammalian transcriptomes by RNA-Seq. NatMethods 5, 621-628

Pandey, R. R., Mondal, T., Mohammad, F., Enroth, S., Redrup, L.,Komorowski, J., Nagano, T., Mancini-Dinardo, D., and Kanduri, C. (2008).Kcnqlotl antisense noncoding RNA mediates lineage-specifictranscriptional silencing through chromatin-level regulation. Mol Cell32, 232-246

Pasini, D., Bracken, A. P., Jensen, M. R., Lazzerini Denchi, E., andHelin, K.

(2004). Suz12 is essential for mouse development and for EZH2 histonemethyltransferase activity. EMBO J 23, 4061-4071

Pasini, D., Cloos, P. A., Walfridsson, J., Olsson, L., Bukowski, J. P.,Johansen, J. V, Bak, M., Tommerup, N., Rappsilber, J., and Helin, K.JARID2 regulates binding of the Polycomb repressive complex 2 to targetgenes in ES cells. Nature 464, 306-310

Peng, J. C., Valouev, A., Swigut, T., Zhang, J., Zhao, Y, Sidow, A., andWysocka, J. (2009). Jarid2/Jumonji Coordinates Control of PRC2 EnzymaticActivity and Target Gene Occupancy in Pluripotent Cells. Cell 139,1290-1302

Pietersen, A. M., and van Lohuizen, M. (2008). Stem cell regulation bypolycomb repressors: postponing commitment. Curr Opin Cell Biol 20,201-207

Rajasekhar, V K., and Begemann, M. (2007). Concise review: roles ofpolycomb group proteins in development and disease: a stem cellperspective. Stem Cells 25, 2498-2510

Ringrose, L., and Paro, R. (2004). Epigenetic regulation of cellularmemory by the Polycomb and Trithorax group proteins. Annu Rev Genet 38,413-443

Rinn, J .L., Kertesz, M., Wang, J. K., Squazzo, S. L., Xu, X., Brugmann,S. A., Goodnough, L. H., Helms, J. A., Farnham, P. J., Segal, E., et al.(2007). Functional demarcation of active and silent chromatin domains inhuman HOX loci by noncoding RNAs. Cell 129, 1311-1323

Schoeftner, S., Sengupta, A. K., Kubicek, S., Mechtler, K., Spahn, L.,Koseki, H., Jenuwein, T., and Wutz, A. (2006). Recruitment of PRC1function at the initiation of X inactivation independent of PRC2 andsilencing. Embo J 25, 3110-3122

Schuettengruber, B., Chourrout, D., Vervoort, M., Leblanc, B., andCavalli, G (2007). Genome regulation by polycomb and trithorax proteins.Cell 128, 735-745

Schwartz, Y. B., Kahn, T. G, Nix, D. A., Li, X. Y, Bourgon, R., Biggin,M., and Pirrotta, V. (2006). Genome-wide analysis of Polycomb targets inDrosophila melanogaster. Nat Genet 38, 700-705

Schwartz, Y. B., and Pirrotta, V. (2008). Polycomb complexes andepigenetic states. Curr Opin Cell Biol 20, 266-273

Seila, A. C., Calabrese, J. M., Levine, S. S., Yeo, G W., Rahl, P. B.,Flynn, R. A., Young, R. A., and Sharp, P. A. (2008). Divergenttranscription from active promoters. Science 322, 1849-1851

Shen, X., Liu, Y., Hsu, Y. J., Fujiwara, Y, Kim, J., Mao, X., Yuan, GC., and Orkin, S. H. (2008). EZH1 mediates methylation on histone H3lysine 27 and complements EZH2 in maintaining stem cell identity andexecuting pluripotency. Mol Cell 32, 491-502

Shen, X., Woojin, K., Fujiwara, Y, Simon, M. D., Liu, Y., Mysliwiec, M.R., Yuan, G C., Lee, Y, and Orkin, S. H. (2009). Jumonji ModulatesPolycomb Activity and Self-Renewal versus Differentiation of Stem Cells.Cell 139, 1303-1314

Simon, J. A., and Lange, C. A. (2008). Roles of the EZH2 histonemethyltransferase in cancer epigenetics. Mutat Res 647, 21-29

Sing, A., Pannell, D., Karaiskakis, A., Sturgeon, K., Djabali, M.,Ellis, J., Lipshitz, H. D., and Cordes, S. P. (2009). A vertebratePolycomb response element governs segmentation of the posteriorhindbrain. Cell 138, 885-897

Sparmann, A., and van Lohuizen, M. (2006). Polycomb silencers controlcell fate, development and cancer. Nat Rev Cancer 6, 846-856

Taft, R. J., Glazov, E. A., Cloonan, N., Simons, C., Stephen, S.,Faulkner, G J., Lassmann, T., Forrest, A. R., Grimmond, S. M., Schroder,K., et al. (2009). Tiny RNAs associated with transcription start sitesin animals. Nat Genet 41, 572-578

Takahashi, N., Okamoto, A., Kobayashi, R., Shirai, M., Obata, Y., Ogawa,H.,

Sotomaru, Y, and Kono, T. (2009). Deletion of Gtl2, imprinted non-codingRNA, with its differentially methylated region induces lethalparent-origin-dependent defects in mice. Hum Mol Genet 18, 1879-1888

Thorvaldsen, J. L., and Bartolomei, M. S. (2007). SnapShot: imprintedgene clusters. Cell 130, 958

Ule, J., Jensen, K., Mele, A., and Darnell, R. B. (2005). CLIP: a methodfor identifying protein-RNA interaction sites in living cells. Methods37, 376-386

Wan, L. B., and Bartolomei, M. S. (2008). Regulation of imprinting inclusters: noncoding RNAs versus insulators. Adv Genet 61, 207-223

Williamson, C. M., Turner, M. D., Ball, S. T., Nottingham, W. T.,Glenister, P., Fray, M., Tymowska-Lalanne, Z., Plagge, A.,Powles-Glover, N., Kelsey, G., et al. (2006). Identification of animprinting control region affecting the expression of all transcripts inthe Gnas cluster. Nat Genet 38, 350-355

Woo, C. J., Kharchenko, P. V, Daheron, L., Park, P. J., and Kingston, R.E. (2010). A region of the human HOXD cluster that confersPolycomb-group responsiveness. Cell 140, 99-110

Yap, K. L., Li, S., Munoz-Cabello, A. M., Raguz, S., Zeng, L., Mujtaba,S., Gil, J., Walsh, M. J., and Zhou, M. M. (2010). Molecular interplayof the noncoding RNA ANRIL and methylated histone H3 lysine 27 bypolycomb CBX7 in transcriptional silencing of INK4a. Mol Cell 38,662-674

Zhao, J., Sun, B. K., Erwin, J. A., Song, J. J., and Lee, J. T. (2008).Polycomb proteins targeted by a short repeat RNA to the mouse Xchromosome. Science 322, 750-756

Prasanth et al., Cell, 123:249-263, 2005

Khalil et al., Proc. Natl. Acad. Sci. USA, 106:11667-1672, 2009

Bernard et al., EMBO J, 29:3082-3093, 2010

Mariner et al., Molec. Cell, 29:499-509, 2008

Shamovsky et al., Nature, 440:556-560, 2006

Sunwoo et al., Genome Res., 19:347-359, 2009

Kanhere et al., Molec. Cell, 38:675-388, 2010

Sarma et al., Proc. Natl. Acad. Sci., USA 107:22196-22201, 2010

Examples of Embodiments

Examples of embodiments described herein include, but are not limitedto:

1. A method of preparing a plurality of validated cDNAs complementary toa pool of nuclear ribonucleic acids (nRNAs), the method comprising:

providing a sample comprising nuclear ribonucleic acids, e.g., a samplecomprising nuclear lysate, e.g., comprising nRNAs bound to nuclearproteins;

contacting the sample with an agent, e.g., an antibody, that bindsspecifically to a nuclear protein that is known or suspected to bind tonuclear ribonucleic acids, e.g., Ezh2, G9a, or Cbx7, under conditionssufficient to form complexes between the agent and the protein, e.g.,such that the nRNAs remain bound to the proteins;

isolating the complexes;

synthesizing DNA complementary to the nRNAs to provide an initialpopulation of cDNAs;

optionally PCR-amplifying the cDNAs using strand-specific primers;

purifying the initial population of cDNAs to obtain a purifiedpopulation of cDNAs that are at least about 20 nucleotides (nt) inlength, e.g., at least 25, 50, 100, 150 or 200 nt in length;

sequencing at least part or substantially all of the purified populationof cDNAs; comparing the high-confidence sequences to a reference genome,and selecting those sequences that have a high degree of identity tosequences in the reference genome, e.g., at least 95%, 98%, or 99%identity, or that have fewer than 10, 5, 2, or 1 mismatches; and

selecting those cDNAs that have (i) reads per kilobase per million reads(RPKM) above a desired threshold, and (ii) are enriched as compared to acontrol library (e.g., a protein-null library or library made from anIgG pulldown done in parallel);

thereby preparing the library of cDNAs.

2. The method of embodiment 1, wherein the agent is an antibody andisolating the complexes comprises immunoprecipitating the complexes.

3. The method of embodiment 1, wherein the cDNAs are synthesized usingstrand-specific adaptors.

4. The method of embodiment 1, further comprising sequencingsubstantially all of the cDNAs. 5. A library of cDNAs complementary to apool of nuclear ribonucleic acids (nRNAs) prepared by the method ofembodiments 1-4.

6. The library of embodiment 5, wherein each of the cDNAs is linked toan individually addressable bead or area on a substrate.

7. An isolated nucleic acid comprising a sequence referred to in Table1, 2, 3, 6, and/or 7, or a fragment comprising at least 20 nt thereof.

8. A method of decreasing expression of an oncogene in a cell, themethod comprising contacting the cell with a long non-coding RNA, orPRC2-binding fragment thereof, as referred to in Table 6 or a nucleicacid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% homologous to a lncRNA sequence, or PRC2-bindingfragment thereof, as referred to in Table 6.

9. The method of embodiment 8, wherein the oncogene is c-myc.

10. The method of embodiment 9, wherein the long non-coding RNA is Pvt1.

11. A method of increasing expression of a tumor suppressor in a mammal,e.g. human, in need thereof comprising administering to said mammal aninhibitory nucleic acid that specifically binds to a human lncRNAcorresponding to a tumor suppressor locus of Table 7, or a human lncRNAcorresponding to an imprinted gene of Table 1, and/or a human lncRNAcorresponding to a growth-suppressing gene of Table 2, or a relatednaturally occurring lncRNA that is orthologous or at least 90%, (e.g.,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical over atleast 15 (e.g., at least 20, 21, 25, 30, 100) nucleobases thereof, in anamount effective to increase expression of the tumor suppressor.

12. A method of inhibiting or suppressing tumor growth in a mammal, e.g.human, with cancer comprising administering to said mammal an inhibitorynucleic acid that specifically binds to a human lncRNA corresponding toa tumor suppressor locus of Table 7, or a human lncRNA corresponding toan imprinted gene of Table 1, and/or a human lncRNA corresponding to agrowth-suppressing gene of Table 2, or a related naturally occurringlncRNA that is orthologous or at least 90%, (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical over at least 15 (e.g., atleast 20, 21, 25, 30, 50, 70, 100) nucleobases thereof, in an amounteffective to suppress or inhibit tumor growth.

13. A method of treating a mammal, e.g., a human, with cancer comprisingadministering to said mammal an inhibitory nucleic acid thatspecifically binds to a human lncRNA corresponding to a tumor suppressorlocus of Table 7, or a human lncRNA corresponding to an imprinted geneof Table 1, and/or a human lncRNA corresponding to a growth-suppressinggene of Table 2, or a related naturally occurring lncRNA that isorthologous or at least 90% (e.g.,91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical over at least 15 (e.g., at least 20, 21,25, 30, 50, 70, 100) nucleobases thereof, in a therapeutically effectiveamount.

14. The method of any of embodiments 11-13 wherein the inhibitorynucleic acid is single stranded or double stranded.

15. The method of any of embodiments 11-14 wherein the inhibitorynucleic acid is an antisense oligonucleotide, LNA, PNA, ribozyme orsiRNA.

16. The method of any of embodiments 11-15 wherein the inhibitorynucleic acid is 5-40 bases in length (e.g., 12-30, 12-28, 12-25).

17. The method of embodiment 14 wherein the inhibitory nucleic acid isdouble stranded and comprises an overhang (optionally 2-6 bases inlength) at one or both termini.

18. The method of any of embodiments 1-17 wherein the inhibitory nucleicacid comprises a sequence of bases at least 80% or 90% complementary to(e.g., at least 5, 10, 15, 20, 25 or 30 bases of, or up to 30 or 40bases of), or comprises a sequence of bases with up to 3 mismatches(e.g., up to 1, or up to 2 mismatches) over 10, 15, 20, 25 or 30 bases.

19. The method of embodiments 8-18, wherein the cell is a cancer cell,e.g., a tumor cell, in vitro or in vivo, e.g., in a subject.

20. The method of embodiments 8-19, wherein the gene is Nkx2-1.

21. The method of embodiment 20, wherein the long non-coding RNA is atmouse Chromosome 12 from bp 57,636,100 to 57,638,250 in the mouse genomeassembly version NCBI37/mm9 (SEQ ID NO: 191,088), or in the human NKX2-1locus at Chromosome 14 from bp 36,988,521 to 36,991,722, in human genomeassembly version, GRCh37/hg19 (SEQ ID NO: 191,087).

22. A method of enhancing pluripotency of a stem cell, the methodcomprising contacting the cell with a long non-coding RNA, orPRC2-binding fragment thereof, as referred to in Table 3 or a nucleicacid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% homologous to a lncRNA sequence, or PRC2-bindingfragment thereof, as referred to in Table 3.

23. A method of enhancing differentiation of a stem cell, the methodcomprising contacting the cell with an inhibitory nucleic acid thatspecifically binds to a long non-coding RNA as referred to in Table 3.

24. The method of embodiments 22 or 23, wherein the stem cell is anembryonic stem cell.

25. The method of embodiments 22 or 23, wherein the stem cell is an iPScell.

26. A sterile composition comprising an inhibitory nucleic acid thatspecifically binds to or is at least 90% complementary to (e.g., atleast 5, 10, 15, 20, 25 or 30 bases of, or up to 30 or 40 bases of) alncRNA of Table 1, 2, 6, or 7, or a related naturally occurring lncRNAat least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to at least 15 (e.g., at least 20, 21, 25, 30, 100)nucleobases of an lncRNA of Table 1, 2, 6, or 7, for parenteraladministration.

27. The composition of embodiment 26, wherein the inhibitory nucleicacid is selected from the group consisting of antisenseoligonucleotides, ribozymes, external guide sequence (EGS)oligonucleotides, siRNA compounds, micro RNAs (miRNAs); small, temporalRNAs (stRNA), and single- or double-stranded RNA interference (RNAi)compounds.

28. The composition of embodiment 26, wherein the RNAi) compound isselected from the group consisting of short interfering RNA (siRNA); ora short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa);and small activating RNAs (saRNAs).

29. The composition of embodiment 26, wherein the antisenseoligonucleotide is selected from the group consisting of antisense RNAs,antisense DNAs, chimeric antisense oligonucleotides, and antisenseoligonucleotides

30. The composition of any of embodiments 26-29, wherein the inhibitorynucleic acid comprises one or more modifications comprising: a modifiedsugar moiety, a modified internucleoside linkage, a modified nucleotideand/or combinations thereof

31. The composition of embodiment 30, wherein the modifiedinternucleoside linkage comprises at least one of: alkylphosphonate,phosphorothioate, phosphorodithioate, alkylphosphonothioate,phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate,carboxymethyl ester, or combinations thereof

32. The composition of embodiment 30, wherein the modified sugar moietycomprises a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxymodified sugar moiety, a 2′-O-alkyl modified sugar moiety, or a bicyclicsugar moiety.

Yet other examples of embodiments include, but are not limited to:

1A. A locked nucleic acid (LNA) molecule that is complementary to andbinds specifically to a long noncoding RNA (lncRNA).

With respect to these embodiments, lncRNA includes endogenous cellularRNAs that are greater than 60 nt in length, e.g., greater than 100 nt,e.g., greater than 200 nt, have no positive-strand open reading framesgreater than 100 amino acids in length, are identified as lncRNAs byexperimental evidence, and are distinct from known (smaller)functional-RNA classes (including but not limited to ribosomal,transfer, and small nuclear/nucleolar RNAs, siRNA, piRNA, and miRNA).See, e.g., Lipovich et al., “MacroRNA underdogs in a microRNA world:Evolutionary, regulatory, and biomedical significance of mammalian longnon-protein-coding RNA” Biochimica et Biophysica Acta (2010)doi:10.1016/j.bbagrm.2010.10.001; Ponting et al., Cell 136(4):629-641(2009), Jia et al., RNA 16 (8) (2010) 1478-1487, Dinger et al., NucleicAcids Res. 37 1685 (2009) D122-D126 (database issue); and referencescited therein. LncRNAs have also been referred to as long RNA, largeRNA, macro RNA, intergenic RNA, and NonCoding Transcripts.

2A. The molecule of embodiment 1A, wherein the lncRNA is a largeintergenic non-coding RNA (lincRNA), a promoter associated short RNA(PASR), an endogenous antisense RNA, or an RNA that binds a chromatinmodifier, e.g., a Polycomb complex, e.g., Polycomb repressive complex 2.

3A. The molecule of embodiment 1A, wherein the lncRNA is localized tothe nucleus. 4A. The molecule of embodiment 1A, wherein the LNA moleculeis complementary to a region of the lncRNA comprising a known RNAlocalization motif.

5A. The method of embodiment 1A, wherein the LNA comprises at least onenon-locked nucleotide.

6A. A method of dissociating a long noncoding RNA (lncRNA) from itscognate binding sequence, the method comprising contacting the lncRNAwith a locked nucleic acid (LNA) molecule that is complementary to andbinds specifically to the lncRNA.

7A. A method of decreasing binding of a long noncoding RNA (lncRNA) toits cognate binding sequence, the method comprising contacting thelncRNA with a locked nucleic acid (LNA) molecule that is complementaryto and binds specifically to the lncRNA.

8A. The method of embodiment 6A or 7A, wherein the lncRNA is a largeintergenic non-coding RNA (lincRNA), a promoter associated short RNA(PASR), an endogenous antisense RNA, or an RNA that binds a chromatinmodifier.

9A. The method of embodiment 6A or 7A, wherein the lncRNA is localizedto the nucleus.

10A. The method of embodiment 6A or 7A, wherein the LNA molecule iscomplementary to a region of the lncRNA comprising a known RNAlocalization motif. 11A. The method of embodiment 6A or 7A, wherein theLNA comprises at least one non-locked nucleotide.

Yet other examples of embodiments include, but are not limited to:

1B. An inhibitory nucleic acid that specifically binds, or iscomplementary to, an RNA that is known to bind to Polycomb repressivecomplex 2 (PRC2), optionally an RNA of SEQ ID NO: 17040, or an RNA ofany of Tables 1-8 or an RNA of any of SEQ ID NOS: 1 to 193,049, for usein the treatment of disease, wherein the treatment involves modulatingexpression of a gene targeted by the RNA, wherein the inhibitory nucleicacid is between 5 and 40 bases in length, and wherein the inhibitorynucleic acid is formulated as a sterile composition.

2B. A process of preparing an inhibitory nucleic acid that specificallybinds, or is complementary to, an RNA that is known to bind to Polycombrepressive complex 2 (PRC2), the process comprising the step ofdesigning and/or synthesizing an inhibitory nucleic acid of between 5and 40 bases in length, optionally single stranded, that specificallybinds to an RNA sequence that binds to PRC2, optionally an RNA of SEQ IDNO: 17040, or an RNA of any of Tables 1-8 or an RNA of any of SEQ IDNOS: 1 to 193,049.

3B. The process of embodiment 2B wherein prior to designing and/orsynthesising the inhibitory nucleic acid the process further comprisesidentifying an RNA that binds to PRC2.

4B. The process of embodiment 2B wherein the RNA has been identified bya method involving identifying an RNA that binds to PRC2.

5B. The process of embodiment 2B, wherein the sequence of the designedand/or synthesised inhibitory nucleic acid is based on a said RNAsequence that binds to PRC2, or a portion thereof, said portion having alength of from 15 to 100 contiguous base pairs.

6B. The process of embodiment 2B, wherein the sequence of the designedand/or synthesised inhibitory nucleic acid is based on a nucleic acidsequence that is complementary to said RNA sequence that binds to PRC2,or is complementary to a portion thereof, said portion having a lengthof from 5 to 40 contiguous base pairs;

7B. The process of any one of embodiments 2B to 6B, wherein theinhibitory nucleic acid is for use in the manufacture of apharmaceutical composition or medicament for use in the treatment ofdisease, optionally wherein the treatment involves modulating expressionof a gene targeted by the RNA binds to PRC2.

8B. A sterile composition comprising an inhibitory nucleic acid thatspecifically binds, or is complementary to, an RNA sequence of any oneof SEQ ID NOS: 1 to 193,049, and is capable of modulating expression ofa gene targeted by the RNA.

9B. An inhibitory nucleic acid for use in the treatment of disease,wherein said inhibitory nucleic acid specifically binds, or iscomplementary to, an RNA sequence of any one of SEQ ID NOS: 1 to193,049, and wherein the treatment involves modulating expression of agene targeted by the RNA.

10B. A method of modulating gene expression comprising administering toa mammal an inhibitory nucleic acid that specifically binds, or iscomplementary to, an RNA sequence of any one of SEQ ID NOS: 1 to193,049, in an amount effective for modulating expression of a genetargeted by the RNA.

11B. An inhibitory nucleic acid for use in the treatment of disease,wherein said inhibitory nucleic acid specifically binds, or iscomplementary to, a mouse RNA sequence of any one of the mouse RNAs ofTables 1-7, e.g., set forth in SEQ ID NOS: 1 to 12,603, and wherein thetreatment involves modulating expression of a gene targeted by the RNA.

12B. An inhibitory nucleic acid for use in the treatment of disease,wherein said inhibitory nucleic acid specifically binds, or iscomplementary to a human RNA sequence corresponding to a mouse RNAsequence of any one of the mouse RNAs of Tables 1-7, e.g., set forth inSEQ ID NOS: 1 to 12,603 wherein the human RNA sequence is (a) obtainableby mapping of highly conserved regions from the mouse to human genome,or by mapping of syntenic positions from the mouse to human genome,e.g., mouse-to-human LiftOver analysis, or (b) is at least 90% identicalover at least 15 bases (or at least 20, 21, 25, 30, or 100 bases) to themouse RNA sequence, and wherein the treatment involves modulatingexpression of a gene targeted by the RNA.

13B. The inhibitory nucleic acid of embodiment 12B wherein the human RNAsequence is any one of the human RNAs of Table 1-7, e.g. set forth inSEQ ID NOS: 12,604 to 21,582 or 191,089 to 193,049.

14B. A method of modulating expression of a gene comprisingadministering to a mammal an inhibitory nucleic acid that specificallybinds, or is complementary to, a mouse RNA sequence of any one of themouse RNAs of Tables 1-7, e.g. set forth in SEQ ID NOS: 1 to 12,603, inan amount effective for modulating expression of a gene targeted by theRNA.

15B. A method of modulating expression of a gene comprisingadministering to a mammal an inhibitory nucleic acid that specificallybinds, or is complementary to a human RNA sequence that corresponds to amouse RNA sequence of any one of the mouse RNAs of Tables 1-7, e.g. setforth in SEQ ID NOS: 1 to 12,603, wherein the human RNA sequence is (a)obtainable by mapping of highly conserved regions from the mouse tohuman genome, or by mapping of syntenic positions from the mouse tohuman genome, e.g., mouse-to-human LiftOver analysis, or (b) is at least90% identical over at least 15 bases (or at least 20, 21, 25, 30, or 100bases) to the mouse RNA sequence, in an amount effective for modulatingexpression of a gene targeted by the RNA. 16B. The method of embodiment15B wherein the human RNA sequence is any one of the human RNAs ofTables 1-7, e.g. set forth in SEQ ID NOS: 12,604 to 21,582 or 191,089 to193,049.

17B. A sterile composition comprising an inhibitory nucleic acid thatspecifically binds, or is complementary to, a human RNA sequence of anyof the human Peaks, e.g. set forth in SEQ ID NOS: 124,437 to 190,716, or190,934 to 191,086, or 191,087, or a mouse RNA sequence of any of themouse Peaks, e.g. set forth in SEQ ID NOS: 21,583 to 124,436, or 190,717to 190,933, or 191,088, and that is capable of modulating expression ofa gene targeted by the RNA.

18B. An inhibitory nucleic acid for use in the treatment of disease,wherein said inhibitory nucleic acid specifically binds, or iscomplementary to, a human RNA sequence of any of the human Peaks, e.g.set forth in SEQ ID NOS: 124,437 to 190,716, or 190,934 to 191,086, or191,087, or a mouse RNA sequence of any of the mouse Peaks, e.g. setforth in SEQ ID NOS: 21,583 to 124,436, or 190,717 to 190,933, or191,088, and wherein the treatment involves modulating expression of agene targeted by the RNA.

19B. A method of modulating expression of a gene comprisingadministering to a mammal an inhibitory nucleic acid that specificallybinds, or is complementary to, a mouse RNA sequence of any of the humanPeaks, e.g. set forth in SEQ ID NOS: 124,437 to 190,716, or 190,934 to191,086, or 191,087, or a mouse RNA sequence of any of the mouse Peaks,e.g. set forth in SEQ ID NOS: 21,583 to 124,436, or 190,717 to 190,933,or 191,088, in an amount effective for modulating expression of a genetargeted by the RNA.

20B. An inhibitory nucleic acid of about 5 to 50 bases in length thatspecifically binds, or is complementary to, a fragment of any of the RNAof SEQ ID NOS: 1 to 21,582 or 191,089 to 193,049, said fragment about2000, 1750, 1500, 1250, 1000, 750, 500, 400, 300, 200, or about 100bases in length (or any range in between any of these numbers), whereinthe fragment of RNA overlaps with and comprises a stretch of at leastfive (5), 10, 15, 20, 25, 30, 35, 40, 45, or 50 consecutive bases withinany of the human Peaks, e.g. set forth in SEQ ID NOS: 124,437 to190,716, or 190,934 to 191,086, or 191,087, or of any of the mousePeaks, e.g. set forth in SEQ ID NOS: 21,583 to 124,436, or 190,717 to190,933, or 191,088, optionally for use in the treatment of disease,wherein the treatment involves modulating expression of a gene targetedby the RNA.

21B. A method of modulating expression of a gene comprisingadministering to a mammal an inhibitory nucleic acid of embodiment 20Bin an amount effective for modulating expression of a gene targeted bythe RNA.

22B. The inhibitory nucleic acid, process, composition or method of anyof the preceding embodiments wherein the modulating is upregulating geneexpression, optionally wherein the gene targeted by the RNA is selectedfrom the group of genes set forth in Table 8, and wherein the RNAsequences are selected from the SEQ ID NOs of the RNAs that target thegene as shown in Table 8.

23B. A sterile composition comprising an inhibitory nucleic acid thatspecifically binds, or is complementary to a mouse RNA sequence of anyone of the mouse RNAs of Tables 1, 2, 6 or 7, e.g. as set forth in SEQID NOS: 1 to 9,836 or 12,053 to 12,603.

24B. A sterile composition comprising an inhibitory nucleic acid thatspecifically binds, or is complementary to, a human RNA sequencecorresponding to a mouse RNA sequence of any one of the mouse RNAs ofTables 1, 2, 6 or 7, e.g. as set forth in SEQ ID NOS: 1 to 9,836, or12,053 to 12,603.

25B. The sterile composition of embodiment 24B wherein (a) the human RNAsequence is obtainable by mapping of highly conserved regions from themouse to human genome, or by mapping of syntenic positions from themouse to human genome, e.g., mouse-to-human LiftOver analysis, or (b)the human RNA sequence is at least 90% identical over at least 15 bases(or at least 20, 21, 25, 30, or 100 bases) to the mouse RNA sequence.

26B. The sterile composition of embodiment 24B wherein the human RNAsequence is any one of the human RNAs of Tables 1, 2, 6 or 7, e.g. asset forth in SEQ ID NOS: 12,604 to 19,236, or 21,195 to 21,582, or191,089 to 192,885, or 192,980 to 193,049.

27B. The sterile composition of any of the preceding embodiments whichis for parenteral administration.

28B. The sterile composition of any of the preceding embodiments whereinthe inhibitory nucleic acid is capable of upregulating expression of agene targeted by the RNA.

29B. A composition for use in a method of increasing expression of atumor suppressor, for use in a method of inhibiting or suppressing tumorgrowth, or for use in a method of treating cancer, the compositioncomprising an inhibitory nucleic acid that specifically binds, or iscomplementary to, a mouse RNA sequence of any one of the mouse RNAs ofTables 1 or 7, e.g. as set forth in SEQ ID NOS: 1 to 49, or 12,268 to12,603, for use in a method of increasing expression of a tumorsuppressor, for use in a method of inhibiting or suppressing tumorgrowth, or for use in a method of treating cancer.

30B. A composition for use in a method of increasing expression of atumor suppressor, for use in a method of inhibiting or suppressing tumorgrowth, or for use in a method of treating cancer, the compositioncomprising an inhibitory nucleic acid that specifically binds, or iscomplementary to, a human RNA sequence orthologous to a mouse RNAsequence of any one of the mouse RNAs of Tables 1 or 7, e.g. as setforth in SEQ ID NOS: 1 to 49, or 12,268 to 12,603.

31B. A method of increasing expression of a tumor suppressor in a mammalin need thereof comprising administering to said mammal an inhibitorynucleic acid that specifically binds, or is complementary to a mouse RNAsequence of any one of the mouse RNAs of Tables 1 or 7, e.g. as setforth in SEQ ID NOS: 1 to 49, or 12,268 to 12,603, in an amounteffective to increase expression of the tumor suppressor.

32B. A method of increasing expression of a tumor suppressor in a mammalin need thereof comprising administering to said mammal an inhibitorynucleic acid that specifically binds, or is complementary to a human RNAsequence corresponding to a mouse RNA sequence of any one of the mouseRNAs of Tables 1 or 7, e.g. as set forth in SEQ ID NOS: 1 to 49, or12,268 to 12,603, in an amount effective to increase expression of thetumor suppressor.

33B. A method of inhibiting or suppressing tumor growth in a mammal inneed thereof comprising administering to said mammal an inhibitorynucleic acid that specifically binds, or is complementary to a mouse RNAsequence of any one of the mouse RNAs of Tables 1 or 7, e.g. as setforth in SEQ ID NOS: 1 to 49, or 12,268 to 12,603, in an amounteffective to suppress or inhibit tumor growth.

34B. A method of inhibiting or suppressing tumor growth in a mammal inneed thereof comprising administering to said mammal an inhibitorynucleic acid that specifically binds, or is complementary to a human RNAsequence corresponding to a mouse RNA sequence of any one of the mouseRNAs of Tables 1 or 7, e.g. as set forth in SEQ ID NOS: 1 to 49, or12,268 to 12,603, in an amount effective to suppress or inhibit tumorgrowth.

35B. A method of treating a mammal with cancer comprising administeringto said mammal an inhibitory nucleic acid that specifically binds, or iscomplementary to a mouse RNA sequence of any one of the mouse RNAs ofTables 1 or 7, e.g. as set forth in SEQ ID NOS: 1 to 49, or 12,268 to12,603, in a therapeutically effective amount.

36B. A method of treating a mammal with cancer comprising administeringto said mammal an inhibitory nucleic acid that specifically binds, or iscomplementary to a human RNA sequence corresponding to a mouse RNAsequence of any one of the mouse RNAs of Tables 1 or 7, e.g. as setforth in SEQ ID NOS: 1 to 49, or 12,268 to 12,603, in a therapeuticallyeffective amount.

37B. The composition or method of any of embodiments 29B-36B wherein (a)the human RNA sequence is obtainable by mapping of highly conservedregions from the mouse to human genome, or by mapping of syntenicpositions from the mouse to human genome, e.g., mouse-to-human LiftOveranalysis, or (b) the human RNA sequence is at least 90% identical overat least 15 bases (or at least 20, 21, 25, 30, or 100 bases) to themouse RNA sequence.

38B. The composition or method of any of embodiments 29B-36B wherein thehuman RNA sequence is any one of the human RNAs of Tables 1 or 7, e.g.as set forth in SEQ ID NOS: 12,604 to 12,632, or 21,338 to 21,582, or192,874 to 192,885 or 193,007 to 193,049.

39B. A method of enhancing differentiation of a stem cell, optionally anembryonic stem cell, and optionally an iPS cell, the method comprisingcontacting the cell with an inhibitory nucleic acid that specificallybinds, or is complementary to, a mouse RNA sequence of any one of themouse RNAs of Table 3, e.g. as set forth in SEQ ID NOS: 9,837 to 10,960.

40B. A method of enhancing differentiation of a stem cell, optionally anembryonic stem cell, and optionally an iPS cell, the method comprisingcontacting the cell with an inhibitory nucleic acid that specificallybinds, or is complementary to, a human RNA sequence corresponding to amouse RNA sequence of any one of the mouse RNAs of Table 3, e.g. as setforth in SEQ ID NOS: 9,837 to 10,960.

41B. The method of embodiment 40B wherein (a) the human RNA sequence isobtainable by mapping of highly conserved regions from the mouse tohuman genome, or by mapping of syntenic positions from the mouse tohuman genome, e.g., mouse-to-human LiftOver analysis, or (b) the humanRNA sequence is at least 90% identical over at least 15 bases (or atleast 20, 21, 25, 30, or 100 bases) to the mouse RNA sequence.

42B. The method of embodiment 40B wherein the corresponding human RNAsequence is any one of the human RNAs of Table 3, e.g. as set forth inSEQ ID NOS: 19,237 to 20,324, or 192,886 to 192,906.

43B. The method of any of embodiments 39B-42B carried out ex vivo,optionally to differentiate the stem cell into a particular cell type,optionally nerve, neuron, dopaminergic neuron, muscle, skin, heart,kidney, liver, lung, neuroendocrine, retinal, retinal pigmentepithelium, pancreatic alpha or beta cells, hematopoietic, chondrocyte,bone cells, blood cells T-cells, B-cells, macrophages, erythrocytes, orplatelets.

44B. The inhibitory nucleic acid, process, composition or method of anyof the preceding embodiments wherein the inhibitory nucleic acid is 5 to40 bases in length (optionally 12-30, 12-28, or 12-25 bases in length).

45B. The inhibitory nucleic acid, process, composition or method of anyof the preceding embodiments wherein the inhibitory nucleic acid is 10to 50 bases in length.

46B. The inhibitory nucleic acid, process, composition or method of anyof the preceding embodiments wherein the inhibitory nucleic acidcomprises a sequence of bases at least 80% or 90% complementary to(including fully complementary to), e.g., at least 5-30, 10-30, 15-30,20-30, 25-30 or 5-40, 10-40, 15-40, 20-40, 25-40, or 30-40 bases of theRNA sequence. It is understood that complementarity is determined over acontiguous stretch of bases, e.g. 5-30 contiguous bases.

47B. The inhibitory nucleic acid, process, composition or method of anyof the preceding embodiments wherein the inhibitory nucleic acidcomprises a sequence of bases at least 90% complementary to at least 10bases of the RNA sequence.

48B. The inhibitory nucleic acid, process, composition or method of anyof the preceding embodiments wherein the inhibitory nucleic acidcomprises a sequence of bases with up to 3 mismatches (e.g., up to 1, orup to 2 mismatches) in complementary base pairing over 10, 15, 20, 25 or30 bases of the RNA sequence.

49B. The inhibitory nucleic acid, process, composition or method of anyof the preceding embodiments wherein the inhibitory nucleic acidcomprises a sequence of bases at least 80% complementary to at least 10bases of the RNA sequence.

50B. The inhibitory nucleic acid, process, composition or method of anyof the preceding embodiments wherein the inhibitory nucleic acidcomprises a sequence of bases with up to 3 mismatches over 15 bases ofthe RNA sequence.

51B. The inhibitory nucleic acid, process, composition or method of anyof the preceding embodiments wherein the inhibitory nucleic acid issingle stranded.

52B. The inhibitory nucleic acid, process, composition or method of anyof the preceding embodiments wherein the inhibitory nucleic acid isdouble stranded.

53B. The inhibitory nucleic acid, process, composition or method of anyof the preceding embodiments wherein the inhibitory nucleic acidcomprises one or more modifications comprising: a modified sugar moiety,a modified internucleoside linkage, a modified nucleotide and/orcombinations thereof.

54B. The inhibitory nucleic acid, process, composition or method of anyof the preceding embodiments wherein the inhibitory nucleic acid is anantisense oligonucleotide, LNA molecule, PNA molecule, ribozyme orsiRNA.

55B. The inhibitory nucleic acid, process, composition or method of anyof the preceding embodiments wherein the inhibitory nucleic acid isdouble stranded and comprises an overhang (optionally 2-6 bases inlength) at one or both termini.

56B. The inhibitory nucleic acid, process, composition or method of anyof the preceding embodiments wherein the inhibitory nucleic acid isselected from the group consisting of antisense oligonucleotides,ribozymes, external guide sequence (EGS) oligonucleotides, siRNAcompounds, micro RNAs (miRNAs); small, temporal RNAs (stRNA), andsingle- or double-stranded RNA interference (RNAi) compounds.

57B. The inhibitory nucleic acid, process, composition or method ofembodiment 56B wherein the RNAi compound is selected from the groupconsisting of short interfering RNA (siRNA); or a short, hairpin RNA(shRNA); small RNA-induced gene activation (RNAa); and small activatingRNAs (saRNAs).

58B. The inhibitory nucleic acid, process, composition or method ofembodiment 54B or 56B wherein the antisense oligonucleotide is selectedfrom the group consisting of antisense RNAs, antisense DNAs, chimericantisense oligonucleotides, and antisense oligonucleotides.

59B. The inhibitory nucleic acid, process, composition or method ofembodiment 53B wherein the modified internucleoside linkage comprises atleast one of: alkylphosphonate, phosphorothioate, phosphorodithioate,alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphatetriester, acetamidate, carboxymethyl ester, or combinations thereof.

60B. The inhibitory nucleic acid, process, composition or method ofembodiment 53B wherein the modified sugar moiety comprises a2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modified sugarmoiety, a 2′-O-alkyl modified sugar moiety, or a bicyclic sugar moiety.

61B. The inhibitory nucleic acid, process, composition or method ofembodiment 53B comprising a 2′-OMe, 2′-F, LNA, PNA, FANA, ENA ormorpholino modification.

62B. A sterile composition comprising an isolated nucleic acid that is amouse RNA sequence of any one of the mouse RNAs of Tables 1-7, e.g. asset forth in SEQ ID

NOS: 1 to 12,603, or a fragment thereof at least 20 bases in length thatretains PRC2-binding activity.

63B. A sterile composition comprising an isolated nucleic acid that is ahuman RNA sequence of any one of the human RNAs of Tables 1-7, e.g. asset forth in SEQ ID NOS: 12,604 to 21,582 or 191,089 to 193,049, or afragment thereof at least 20 bases in length that retains PRC2-bindingactivity.

64B. An RNA for use in a method of decreasing expression of an oncogene,comprising a mouse RNA sequence of any one of the mouse RNAs of Table 6,e.g. as set forth in SEQ ID NOS: 12,053 to 12,267 or a correspondinghuman RNA sequence optionally having a nucleobase sequence of any one ofSEQ ID NOS: 21,195 to 21,337, or 192,980 to 193,006 or a fragmentthereof at least 20 bases in length that retains PRC2-binding activity.

65B. A method of decreasing expression of an oncogene in a cell, themethod comprising contacting the cell with a mouse RNA sequence of anyone of the mouse RNAs of Table 6, e.g. as set forth in SEQ ID NOS:12,053 to 12,267 or a corresponding human RNA sequence optionally havinga nucleobase sequence of any one of SEQ ID NOS: 21,195 to 21,337, or192,980 to 193,006 (see Table 6), or a fragment thereof at least 20bases in length that retains PRC2-binding activity.

66B. An RNA for use in a method of enhancing pluripotency of a stemcell, optionally an embryonic stem cell, and optionally an iPS cell,comprising a mouse RNA sequence of any one of the mouse RNAs of Table 3,e.g. as set forth in SEQ ID NOS: 9,837 to 10,960 or a correspondinghuman RNA sequence optionally having a nucleobase sequence of any one ofSEQ ID NOS: 19,237 to 20,324 or 192,886 to 192,906 (see Table 3), or afragment thereof at least 20 bases in length that retains PRC2-bindingactivity.

67B. A method of enhancing pluripotency of a stem cell, optionally anembryonic stem cell, and optionally an iPS cell, the method comprisingcontacting the cell with a mouse RNA sequence of any one of the mouseRNAs of Table 3, e.g. as set forth in SEQ ID NOS: 9,837 to 10,960 or acorresponding human RNA sequence optionally having a nucleobase sequenceof any one of SEQ ID NOS: 19,237 to 20,324 or 192,886 to 192,906 (seeTable 3), or a fragment thereof at least 20 bases in length that retainsPRC2-binding activity.

68B. A LNA molecule that is complementary to and binds specifically toan lncRNA that binds to a chromatin modifier.

69B. The LNA molecule of embodiment 68B, wherein the chromatin modifieris Polycomb repressive complex 2.

70B. A method of decreasing binding of a long noncoding RNA (lncRNA) toits cognate binding sequence, e.g. PRC2 or a chromosome, the methodcomprising contacting the lncRNA with a locked nucleic acid (LNA)molecule that is complementary to and binds specifically to the lncRNA.

71B. A LNA molecule that is complementary to and binds specifically toan lncRNA that is a large intergenic non-coding RNA (lincRNA), apromoter associated short RNA (PASR), an endogenous antisense RNA, or anRNA that binds a chromatin modifier, e.g., a Polycomb complex, e.g.,Polycomb repressive complex 2.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An inhibitory nucleic acid that specifically binds, or iscomplementary to, an RNA that is known to bind to Polycomb repressivecomplex 2 (PRC2), optionally an RNA of SEQ ID NO: 17040, or an RNA ofany of Tables 1-8 or an RNA of any of SEQ ID NOs: 1 to 193,049, for usein the treatment of disease, wherein the treatment involves modulatingexpression of a gene targeted by the RNA, wherein the inhibitory nucleicacid is between 5 and 40 bases in length, and wherein the inhibitorynucleic acid is formulated as a sterile composition.
 2. A process ofpreparing the inhibitory nucleic acid of claim 1 that binds an RNA thatbinds to Polycomb repressive complex 2 (PRC2), the process comprisingthe step of designing and/or synthesizing a nucleic acid of between 5and 40 bases in length, wherein the RNA is SEQ ID NO: 17040, or an RNAof any of Tables 1-8 or an RNA of any of SEQ ID NOS: 1 to 193,049,wherein the inhibitory nucleic acid is complementary to at least .
 3. Amethod of increasing gene expression of a target gene comprisingadministering to a mammal the inhibitory nucleic acid of claim 1, in anamount effective to increase expression of the gene targeted by the RNA.4. The method of claim 2, wherein the target gene is an oncogene ortumor suppressor, and the inhibitory nucleic acid specifically binds, oris complementary to, an RNA of Table 6 or Table
 7. 5. A method ofinhibiting or suppressing tumor growth in a mammal in need thereofcomprising administering to said mammal an inhibitory nucleic acid thatbinds, or is complementary to a human RNA sequence corresponding to amouse RNA sequence of any of SEQ ID NOs: 1 to 49, or 12,268 to 12,603 inan amount effective to suppress or inhibit tumor growth.
 6. A method ofdecreasing expression of an oncogene in a cell, the method comprisingcontacting the cell with a mouse RNA sequence of any one of SEQ ID NOS:12,053 to 12,267 or a corresponding human RNA sequence optionally havinga nucleobase sequence of any one of SEQ ID NOS: 21,195 to 21,337, or192,980 to 193,006, or a fragment thereof at least 20 bases in lengththat retains PRC2-binding activity.